Promoters for expressing genes in a fungal cell

ABSTRACT

The present invention relates to methods for producing a polypeptide, comprising: (a) cultivating a fungal host cell in a medium conducive for the production of the polypeptide, wherein the fungal host cell comprises a first nucleic acid sequence encoding the polypeptide operably linked to a second nucleic acid sequence comprising a promoter foreign to the nucleic acid sequence, wherein the promoter comprises a sequence selected from the group consisting of nucleotides 1 to 3949 of SEQ ID NO:1, nucleotides 1 to 938 of SEQ ID NO:2, and nucleotides 1 to 3060 of SEQ ID NO:3, and a subsequence thereof; and mutant, hybrid, and tandem promoters thereof; and (b) isolating the polypeptide from the cultivation medium. The present invention also relates to the isolated promoter sequences and to constructs, vectors, and fungal host cells comprising the promoter sequences operably linked to nucleic acid sequences encoding polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.09/534,407, filed Mar. 22, 2000, now U.S. Pat. No. 6,361,973 which is acontinuation-in-part of U.S. application Ser. No. 09/274,449, filed Mar.22, 1999, now abandoned, and claims priority from U.S. provisionalapplication Serial No. 60/145,339, filed Jul. 22, 1999, whichapplications are fully incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to methods for producing polypeptides. Thepresent invention also relates to isolated promoters and to nucleic acidconstructs, vectors, and host cells comprising the promoters operablylinked to nucleic acid sequences encoding polypeptides.

2. Description of the Related Art

The recombinant production of a heterologous protein in a fungal hostcell, particularly a filamentous fungal cell such as Aspergillus, mayprovide for a more desirable vehicle for producing the protein incommercially relevant quantities.

Recombinant production of a heterologous protein is accomplished byconstructing an expression cassette in which the DNA coding for theprotein is placed under the expression control of a promoter, excisedfrom a regulated gene, suitable for the host cell. The expressioncassette is introduced into the host cell, usually by plasmid-mediatedtransformation. Production of the heterologous protein is then achievedby culturing the transformed host cell under inducing conditionsnecessary for the proper functioning of the promoter contained on theexpression cassette.

The development of a new fungal host cell for the recombinant productionof proteins generally requires the availability of promoters that aresuitable for controlling the expression of the proteins in the hostcell. Fusarium venenatum has been shown to be useful as a new host cellfor such expression (WO 96/00787, WO 97/26330). Moreover, the promoterfrom the Fusariuin oxysporum trypsin-like protease gene has beendescribed which is useful for expressing heterologous genes in Fusariumvenenatum host cells (U.S. Pat. No. 5,837,847).

However, there is a need in the art for new promoters for controllingthe expression of heterologous genes.

It is an object of the present invention to provide improved methods forproducing a polypeptide in a fungal host cell and new promoters for suchproduction.

SUMMARY OF THE INVENTION

The present invention relates to methods for producing a polypeptide,comprising: (a) cultivating a fungal host cell in a medium conducive forthe production of the polypeptide, wherein the fungal host cellcomprises a first nucleic acid sequence encoding the polypeptideoperably linked to a second nucleic acid sequence comprising a promoterforeign to the nucleic acid sequence, wherein the promoter comprises asequence selected from the group consisting of nucleotides 1 to 3949 ofSEQ ID NO:1, nucleotides 1 to 938 of SEQ ID NO:2, and nucleotides 1 to3060 of SEQ ID NO:3, and subsequences thereof; and mutant, hybrid, andtandem promoters thereof; and (b) isolating the polypeptide from thecultivation medium.

The present invention also relates to isolated promoters sequences andto constructs, vectors, and fungal host cells comprising one or more ofthe promoters operably linked to a nucleic acid sequence encoding apolypeptide.

The present invention also relates to methods for obtaining mutantpromoters of nucleotides 1 to 3949 of SEQ ID NO:1, nucleotides 1 to 938of SEQ ID NO:2, and nucleotides 1 to 3060 of SEQ ID NO:3.

The present invention also relates to isolated nucleic acid sequences,selected from the group consisting of:

(a) a nucleic acid sequence encoding a polypeptide having an amino acidsequence which has at least 65% identity with amino acids 22 to 581 ofSEQ ID NO:4, at least 65% identity with amino acids 19 to 200 of SEQ IDNO:5, or at least 65% identity with amino acids 1 to 187 of SEQ ID NO:6;

(b) a nucleic acid sequence having at least 65% homology withnucleotides 4013 to 5743 of SEQ ID NO: 1, at least 65% homology withnucleotides 993 to 1593 of SEQ ID NO:2, or at least 65% homology withnucleotides 3061 to 3678 of SEQ ID NO:3;

(c) a nucleic acid sequence which hybridizes under very low, low,medium, medium-high, high, or very high stringency conditions with (i)nucleotides 4013 to 5743 of SEQ ID NO:1, nucleotides 993 to 1593 of SEQID NO:2, or nucleotides 3061 to 3698 of SEQ ID NO:3; (ii) the cDNAsequence contained in nucleotides 4013 to 5743 of SEQ ID NO:1,nucleotides 993 to 1593 of SEQ ID NO:2, or nucleotides 3061 to 3698 ofSEQ ID NO:3; (iii) a subsequence of (i) or (ii) of at least 100nucleotides, or (iv) a complementary strand of (i), (ii), or (iii);

(d) a nucleic acid sequence encoding a variant of the polypeptide havingan amino acid sequence of SEQ ID NO:4, SEQ ID. NO. 5, or SEQ ID NO:6comprising a substitution, deletion, and/or insertion of one or moreamino acids;

(e) an allelic variant of (a), (b), or (c); and

(f) a subsequence of (a), (b), (c), or (e).

The present invention further relates to constructs, vectors, andrecombinant host cells of the nucleic-acid sequences.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1F show the genomic DNA sequence and the deduced amino acidsequence of a Fusarium venenatum glucoamylase gene (SEQ ID NOs:1 and 4,respectively).

FIGS. 2A-2C show the genomic DNA sequence and the deduced amino acidsequence of a Fusarium venenatum gene designated “Daria” (SEQ ID NOs:2and 5, respectively).

FIGS. 3A-3D show the genomic DNA sequence and the deduced amino acidsequence of a Fusarium venenatum gene designated “Quinn” (SEQ ID NOs:3and 6, respectively).

FIG. 4 shows a restriction map of pDM181.

FIG. 5 shows a restriction map of pSheB1.

FIG. 6 shows a restriction map of pDM194.

FIG. 7 shows a restriction map of pJRoy35.

FIG. 8 shows a restriction map of pDM218.

FIG. 9 shows a restriction map of pEJG25A.

FIG. 10 shows a restriction map of pMWR60.

FIG. 11 shows a restriction map of pRaMB62.

FIG. 12 shows a restriction map of pRaMB64.

FIG. 13 shows a restriction map of pRaMB66.

FIG. 14 shows comparative expression of a Humicola lanuginosa lipasereporter gene in Fusarium venenatum under control of Fusarium venenatumamyloglucosidase (pAMG) and Fusarium oxysporum trypsin (pSP387)promoters.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods for producing a polypeptide,comprising (a) cultivating a fungal host cell in a medium conducive forthe production of the polypeptide, wherein the fungal host cellcomprises a first nucleic acid sequence encoding the polypeptideoperably linked to a second nucleic acid sequence comprising a promoterforeign to the first nucleic acid sequence, wherein the promotercomprises a sequence selected from the group consisting of nucleotides 1to 3949 of SEQ ID NO:1, nucleotides 1 to 938 of SEQ ID NO:2, andnucleotides 1 to 3060 of SEQ ID NO:3, and subsequences thereof; andmutant, hybrid, and tandem promoters thereof; and (b) isolating thepolypeptide from the cultivation medium.

In the production methods of the present invention, the cells arecultivated in a nutrient medium suitable for production of thepolypeptide using methods known in the art. For example, the cell may becultivated by shake flask cultivation, small-scale or large-scalefermentation (including continuous, batch, fed-batch, or solid statefermentations) in laboratory or industrial fermentors performed in asuitable medium and under conditions allowing the polypeptide to beexpressed and/or isolated. The cultivation takes place in a suitablenutrient medium comprising carbon and nitrogen sources and inorganicsalts, using procedures known in the art. Suitable media are availablefrom commercial suppliers or may be prepared according to publishedcompositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

The polypeptides may be detected using methods known in the art that arespecific for the polypeptides. These detection methods may include useof specific antibodies, formation of an enzyme product, or disappearanceof an enzyme substrate.

The resulting polypeptide may be recovered by methods known in the art.For example, the polypeptide may be recovered from the nutrient mediumby conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The polypeptides may be purified by a variety of procedures known in theart including, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, J.-C. Jansonand Lars Ryden, editors, VCH Publishers, New York, 1989).

Promoters

The term “promoter” is defined herein as a DNA sequence that binds RNApolymerase and directs the polymerase to the correct downstreamtranscriptional start site of a nucleic acid sequence encoding apolypeptide to initiate transcription. RNA polymerase effectivelycatalyzes the assembly of messenger RNA complementary to the appropriateDNA strand of the coding region. The term “promoter” will also beunderstood to include the 5′ non-coding region (between promoter andtranslation start) for translation after transcription into mRNA,cis-acting transcription control elements such as enhancers, and othernucleotide sequences capable of interacting with transcription factors.

The term “mutant promoter” is defined herein as a promoter having anucleotide sequence comprising a substitution, deletion, and/orinsertion of one or more nucleotides of a parent promoter, wherein themutant promoter has more or less promoter activity than thecorresponding parent promoter. The term “mutant promoter” will alsoencompass natural variants and in vitro generated variants obtainedusing methods well known in the art such as classical mutagenesis,site-directed mutagenesis, and DNA shuffling.

The term “hybrid promoter” is defined herein as parts of two morepromoters that are fused together to generate a sequence that is afusion of the two or more promoters, which is operably linked to acoding sequence and mediates the transcription of the coding sequenceinto mRNA.

The term “tandem promoter” is defined herein as two or more promotersequences each of which is operably linked to a coding sequence andmediates the transcription of the coding sequence into mRNA.

The term “operably linked” is defined herein as a configuration in whicha control sequence, e.g., a promoter sequence, is appropriately placedat a position relative to a coding sequence such that the controlsequence directs the production of a polypeptide encoded by the codingsequence.

The term “coding sequence” is defined herein as a nucleic acid sequencethat is transcribed into mRNA which is translated into a polypeptidewhen placed under the control of the appropriate control sequences. Theboundaries of the coding sequence are generally determined by the ATGstart codon located just upstream of the open reading frame at the 5′end of the mRNA and a transcription terminator sequence located justdownstream of the open reading frame at the 3′ end of the mRNA. A codingsequence can include, but is not limited to, genomic DNA, cDNA,semisynthetic, synthetic, and recombinant nucleic acid sequences.

In a preferred embodiment, the promoter has the nucleic acid sequence ofnucleotides 1 to 3949 of SEQ ID NO:1, or a subsequence thereof. Thesubsequence preferably contains at least about 2100 nucleotides, morepreferably at least about 2400 nucleotides, and most preferably at leastabout 2700 nucleotides.

In another preferred embodiment, the promoter has the nucleic acidsequence of nucleotides 1 to 938 of SEQ ID NO:2, or a subsequencethereof. The subsequence preferably contains at least about 840nucleotides, more preferably at least about 870 nucleotides, and mostpreferably at least about 900 nucleotides.

In another preferred embodiment, the promoter has the nucleic acidsequence of nucleotides 1 to 3060 of SEQ ID NO:3, or a subsequencethereof. The subsequence preferably contains at least about 2100nucleotides, more preferably at least about 2400 nucleotides, and mostpreferably at least about 2700 nucleotides.

In another preferred embodiment, the promoter is the nucleic acidsequence contained in plasmid pECO3 which is contained in Escherichiacoli NRRL B-30067. In another preferred embodiment, the promoter is thenucleic acid sequence contained in plasmid pFAMG which is contained inEscherichia coli NRRL B-30071. In another a preferred embodiment, thepromoter is the nucleic acid contained in plasmid pQUINN which iscontained in Escherichia coli NRRL B-30075.

In the methods of the present invention, the promoter may also be amutant of the promoters described above having a substitution, deletion,and/or insertion of one or more nucleotides in the nucleic acid sequenceof nucleotides 1 to 3949 of SEQ ID NO:1, nucleotides 1 to 938 of SEQ IDNO:2, or nucleotides 1 to 3060 of SEQ ID NO:3.

A mutant promoter may have one or more mutations. Each mutation is anindependent substitution, deletion, and/or insertion of a nucleotide.The introduction of a substitution, deletion, and/or insertion of anucleotide into the promoter may be accomplished using any of themethods known in the art such as classical mutagenesis, site-directedmutagenesis, or DNA shuffling. Particularly useful is a procedure whichutilizes a supercoiled, double stranded DNA vector with an insert ofinterest and two synthetic primers containing the desired mutation. Theoligonucleotide primers, each complementary to opposite strands of thevector, extend during temperature cycling by means of Pfu DNApolymerase. On incorporation of the primers, a mutated plasmidcontaining staggered nicks is generated. Following temperature cycling,the product is treated with DpnI which is specific for methylated andhemimethylated DNA to digest the parental DNA template and to select formutation-containing synthesized DNA. Other procedures known in the artmay also be used.

In a preferred embodiment, the promoter is a mutant of nucleotides 1 to3949 of SEQ ID NO:1. In another preferred embodiment, the promoter is amutant of nucleotides 1 to 938 of SEQ ID NO:2. In another preferredembodiment, the promoter is a mutant of nucleotides 1 to 3060 of SEQ IDNO:3.

In the methods of the present invention, the promoter may also be ahybrid promoter comprising a portion of one or more promoters of thepresent invention; a portion of a promoter of the present invention anda portion of another promoter, e.g., a leader sequence of one promoterand the transcription start site from the other promoter; or a portionof one or more promoters of the present invention and a portion of oneor more other promoters. The other promoter may be any promoter sequencewhich shows transcriptional activity in the fungal host cell of choiceincluding a mutant, truncated, and hybrid promoter, and may be obtainedfrom genes encoding extracellular or intracellular polypeptides eitherhomologous or heterologous to the host cell. The other promoter sequencemay be native or foreign to the nucleic acid sequence encoding thepolypeptide and native or foreign to the cell.

Examples of other promoters useful in the construction of hybridpromoters with the promoters of the present invention include thepromoters obtained from the genes for Aspergillus oryzae TAKA amylase,Rhizoinucor miehei aspartic proteinase, Aspergillus niger neutralalpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillusniger or Aspergillus awamori glucoamylase (glaA), Rhizomucor mieheilipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triosephosphate isomerase, Aspergillus nidulans acetamidase, and Fusariumoxysporum trypsin-like protease (WO 96/00787), as well as the NA2-tpipromoter (a hybrid of the promoters from the genes for Aspergillus nigerneutral alpha-amylase and Aspergillus oryzae triose phosphateisomerase), Saccharomyces cerevisiae enolase (ENO-1), Saccharomycescerevisiae galactokinase (GALI), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP), andSaccharomyces cerevisiae 3-phosphoglycerate kinase, and mutant,truncated, and hybrid promoters thereof. Other useful promoters foryeast host cells are described by Romanos et al., 1992, Yeast 8:423-488.

The promoter may also be a tandem promoter comprising two more promotersof the present invention or alternatively one or more promoters of thepresent invention and one or more other promoters, such as thoseexemplified above. The two or more promoter sequences of the tandempromoter may simultaneously promote the transcription of the nucleicacid sequence. Alternatively, one or more of the promoter sequences ofthe tandem promoter may promote the transcription of the nucleic acidsequence at different stages of growth of the cell.

In the methods of the present invention, the promoter is foreign to thenucleic acid sequence encoding a polypeptide of interest, but thepromoter or nucleic acid sequence may be native to the fungal host cell.A mutant, hybrid, or tandem promoter of the present invention will beunderstood to be foreign to a nucleic acid sequence encoding apolypeptide even if the wild-type promoter is native to the nucleic acidsequence.

A mutant, hybrid, or tandem promoter of the present invention has atleast about 20%, preferably at least about 40%, more preferably at leastabout 60%, more preferably at least about 80%, more preferably at leastabout 90%, more preferably at least about 100%, even more preferably atleast about 200%, most preferably at least about 300%, and even mostpreferably at least about 400% of the promoter activity of the promoterof nucleotides 1 to 3949 of SEQ ID NO:1, nucleotides 1 to 938 of SEQ IDNO:2, or nucleotides 1 to 3060 of SEQ ID NO:3.

Polypeptide Encoding Nucleic Acid Sequences

The polypeptide encoded by the nucleic acid sequence may be native orheterologous to the fungal host cell of interest.

The term “polypeptide” is not meant herein to refer to a specific lengthof the encoded product and, therefore, encompasses peptides,oligopeptides, and proteins. The term “heterologous polypeptide” isdefined herein as a polypeptide which is not native to the fungal cell,a native polypeptide in which modifications have been made to alter thenative sequence, or a native polypeptide whose expression isquantitatively altered as a result of a manipulation of the fungal cellby recombinant DNA techniques. For example, a native polypeptide may berecombinantly produced by, e.g., placing a gene encoding the polypeptideunder the control of a promoter of the present invention to enhanceexpression of the polypeptide, to expedite export of a nativepolypeptide of interest outside the cell by use of a signal sequence,and to increase the copy number of a gene encoding the polypeptidenormally produced by the cell. The fungal cell may contain one or morecopies of the nucleic acid sequence encoding the polypeptide.

Preferably, the polypeptide is a hormone or variant thereof, enzyme,receptor or portion thereof, antibody or portion thereof, or reporter.In a preferred embodiment, the polypeptide is secreted extracellularly.In a more preferred embodiment, the polypeptide is an oxidoreductase,transferase, hydrolase, lyase, isomerase, or ligase. In an even morepreferred embodiment, the polypeptide is an aminopeptidase, amylase,carbohydrase, carboxypeptidase, catalase, cellulase, chitinase,cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase,alpha-galactosidase, beta-galactosidase, glucoamylase,alpha-glucosidase, beta-glucosidase, invertase, laccase, lipase,mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase,phospholipase, phytase, polyphenoloxidase, proteolytic enzyme,ribonuclease, transglutaminase, or xylanase.

The nucleic acid sequence encoding a polypeptide of interest may beobtained from any prokaryotic, eukaryotic, or other source. For purposesof the present invention, the term “obtained from” as used herein inconnection with a given source shall mean that the polypeptide isproduced by the source or by a cell in which a gene from the source hasbeen inserted.

The techniques used to isolate or clone a nucleic acid sequence encodinga polypeptide of interest are known in the art and include isolationfrom genomic DNA, preparation from cDNA, or a combination thereof. Thecloning of the nucleic acid sequence from such genomic DNA can beeffected, e.g., by using the well known polymerase chain reaction (PCR).See, for example, Innis et al., 1990, PCR Protocols. A Guide to Methodsand Application, Academic Press, New York. The cloning procedures mayinvolve excision and isolation of a desired nucleic acid fragmentcomprising the nucleic acid sequence encoding the polypeptide, insertionof the fragment into a vector molecule, and incorporation of therecombinant vector into the mutant fungal cell where multiple copies orclones of the nucleic acid sequence will be replicated. The nucleic acidsequence may be of genomic, cDNA, RNA, semisynthetic, synthetic origin,or any combinations thereof.

In the methods of the present invention, the polypeptide may alsoinclude a fused or hybrid polypeptide in which another polypeptide isfused at the N-terminus or the C-terminus of the polypeptide or fragmentthereof. A fused polypeptide is produced by fusing a nucleic acidsequence (or a portion thereof) encoding one polypeptide to a nucleicacid sequence (or a portion thereof) encoding another polypeptide.Techniques for producing fusion polypeptides are known in the art, andinclude, ligating the coding sequences encoding the polypeptides so thatthey are in frame and expression of the fused polypeptide is undercontrol of the same promoter(s) and terminator. The hybrid polypeptidemay comprise a combination of partial or complete polypeptide sequencesobtained from at least two different polypeptides wherein one or moremay be heterologous to the mutant fungal cell.

Nucleic Acid Constructs

The present invention also relates nucleic acid constructs comprising anucleic acid sequence encoding a polypeptide operably linked to apromoter of the present invention and one or more control sequenceswhich direct the expression of the coding sequence in a suitable hostcell under conditions compatible with the control sequences. Expressionwill be understood to include any step involved in the production of thepolypeptide including, but not limited to, transcription,post-transcriptional modification, translation, post-translationalmodification, and secretion.

“Nucleic acid construct” is defined herein as a nucleic acid molecule,either single- or double-stranded, which is isolated from a naturallyoccurring gene or which has been modified to contain segments of nucleicacid combined and juxtaposed in a manner that would not otherwise existin nature. The term nucleic acid construct is synonymous with the termexpression cassette when the nucleic acid construct contains a codingsequence and all the control sequences required for expression of thecoding sequence.

An isolated nucleic acid sequence encoding a polypeptide may be furthermanipulated in a variety of ways to provide for expression of thepolypeptide. Manipulation of the nucleic acid sequence prior to itsinsertion into a vector may be desirable or necessary depending on theexpression vector. The techniques for modifying nucleic acid sequencesutilizing recombinant DNA methods are well known in the art.

In the methods of the present invention, the nucleic acid sequence maycomprise one or more native control sequences or one or more of thenative control sequences may be replaced with one or more controlsequences foreign to the nucleic acid sequence for improving expressionof the coding sequence in a host cell.

The term “control sequences” is defined herein to include all componentswhich are necessary or advantageous for the expression of a polypeptideof the present invention. Each control sequence may be native or foreignto the nucleic acid sequence encoding the polypeptide. Such controlsequences include, but are not limited to, a leader, polyadenylationsequence, propeptide sequence, promoter of the present invention, signalpeptide sequence, and transcription terminator. At a minimum, thecontrol sequences include a promoter of the present invention, andtranscriptional and translational stop signals. The control sequencesmay be provided with linkers for the purpose of introducing specificrestriction sites facilitating ligation of the control sequences withthe coding region of the nucleic acid sequence encoding a polypeptide.

The control sequence may be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleic acid sequence encoding the polypeptide. Anyterminator which is functional in the host cell of choice may be used inthe present invention.

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus oryzae TAKA amylase, Aspergillus nigerglucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillusniger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA which is important for translation bythe host cell. The leader sequence is operably linked to the 5′ terminusof the nucleic acid sequence encoding the polypeptide. Any leadersequence that is functional in the host cell of choice may be used inthe present invention.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleic acid sequence andwhich, when transcribed, is recognized by the host cell as a signal toadd polyadenosine residues to transcribed mRNA. Any polyadenylationsequence which is functional in the host cell of choice may be used inthe present invention.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, Fusarium oxysporum trypsin-like protease, and Aspergillusniger alpha-glucosidase.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatcodes for an amino acid sequence linked to the amino terminus of apolypeptide and directs the encoded polypeptide into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleic acidsequence may inherently contain a signal peptide coding region naturallylinked in translation reading frame with the segment of the codingregion which encodes the secreted polypeptide. Alternatively, the 5′ endof the coding sequence may contain a signal peptide coding region whichis foreign to the coding sequence. The foreign signal peptide codingregion may be required where the coding sequence does not naturallycontain a signal peptide coding region. Alternatively, the foreignsignal peptide coding region may simply replace the natural signalpeptide coding region in order to enhance secretion of the polypeptide.However, any signal peptide coding region which directs the expressedpolypeptide into the secretory pathway of a host cell of choice may beused in the present invention.

Effective signal peptide coding regions for filamentous fungal hostcells are the signal peptide coding regions obtained from the genes forAspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase,Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase,Humicola insolens cellulase, and Humicola lanuginosa lipase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding regions are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding region that codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to a mature active polypeptideby catalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region may be obtained from thegenes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilisneutral protease (nprT), Saccharomyces cerevisiae alpha-factor,Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophilalaccase (WO 95/33836).

Where both signal peptide and propeptide regions are present at theamino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of a polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion.

It may also be desirable to add regulatory sequences which allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems include the lac,tac, and trp operator systems. In yeast, the ADH2 system or GAL1 systemmay be used. In filamentous fungi, the TAKA alpha-amylase promoter,Aspergillus niger glucoamylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used as regulatory sequences. Otherexamples of regulatory sequences are those which allow for geneamplification. In eukaryotic systems, these include the dihydrofolatereductase gene which is amplified in the presence of methotrexate, andthe metallothionein genes which are amplified with heavy metals. Inthese cases, the nucleic acid sequence encoding the polypeptide would beoperably linked with the regulatory sequence.

The present invention also relates to nucleic acid constructs foraltering the expression of a gene encoding a polypeptide which isendogenous to a host cell. The constructs may contain the minimal numberof components necessary for altering expression of the endogenous gene.In one embodiment, the nucleic acid constructs preferably contain (a) atargeting sequence, (b) a promoter of the present invention, (c) anexon, and (d) a splice-donor site. Upon introduction of the nucleic acidconstruct into a cell, the construct inserts by homologous recombinationinto the cellular genome at the endogenous gene site. The targetingsequence directs the integration of elements (a)-(d) into the endogenousgene such that elements (b)-(d) are operably linked to the endogenousgene. In another embodiment, the nucleic acid constructs contain (a) atargeting sequence, (b) a promoter of the present invention, (c) anexon, (d) a splice-donor site, (e) an intron, and (f) a splice-acceptorsite, wherein the targeting sequence directs the integration of elements(a)-(f) such that elements (b)-(f) are operably linked to the endogenousgene. However, the constructs may contain additional components such asa selectable marker.

In both embodiments, the introduction of these components results inproduction of a new transcription unit in which expression of theendogenous gene is altered. In essence, the new transcription unit is afusion product of the sequences introduced by the targeting constructsand the endogenous gene. In one embodiment in which the endogenous geneis altered, the gene is activated. In this embodiment, homologousrecombination is used to replace, disrupt, or disable the regulatoryregion normally associated with the endogenous gene of a parent cellthrough the insertion of a regulatory sequence which causes the gene tobe expressed at higher levels than evident in the corresponding parentcell. The activated gene can be further amplified by the inclusion of anamplifiable selectable marker gene in the construct using methods wellknown in the art (see, for example, U.S. Pat. No. 5,641,670). In anotherembodiment in which the endogenous gene is altered, expression of thegene is reduced.

The targeting sequence can be within the endogenous gene, immediatelyadjacent to the gene, within an upstream gene, or upstream of and at adistance from the endogenous gene. One or more targeting sequences canbe used. For example, a circular plasmid or DNA fragment preferablyemploys a single targeting sequence, while a linear plasmid or DNAfragment preferably employs two targeting sequences.

The constructs further contain one or more exons of the endogenous gene.An exon is defined as a DNA sequence which is copied into RNA and ispresent in a mature mRNA molecule such that the exon sequence isin-frame with the coding region of the endogenous gene. The exons can,optionally, contain DNA which encodes one or more amino acids and/orpartially encodes an amino acid. Alternatively, the exon contains DNAwhich corresponds to a 5′ non-encoding region. Where the exogenous exonor exons encode one or more amino acids and/or a portion of an aminoacid, the nucleic acid construct is designed such that, upontranscription and splicing, the reading frame is in-frame with thecoding region of the endogenous gene so that the appropriate readingframe of the portion of the mRNA derived from the second exon isunchanged.

The splice-donor site of the constructs directs the splicing of one exonto another exon. Typically, the first exon lies 5′ of the second exon,and the splice-donor site overlapping and flanking the first exon on its3′ side recognizes a splice-acceptor site flanking the second exon onthe 5′ side of the second exon. A splice-acceptor site, like asplice-donor site, is a sequence which directs the splicing of one exonto another exon. Acting in conjunction with a splice-donor site, thesplicing apparatus uses a splice-acceptor site to effect the removal ofan intron.

The present invention further relates to methods for producing apolypeptide comprising (a) cultivating a homologously recombinant cell,having incorporated therein a new transcription unit comprising apromoter of the present invention, an exon, and/or a splice donor siteoperably linked to a second exon of an endogenous nucleic acid sequenceencoding the polypeptide, under conditions conducive for production ofthe polypeptide; and (b) recovering the polypeptide. The methods arebased on the use of gene activation technology, for example, asdescribed in U.S. Pat. No. 5,641,670.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a promoter of the present invention, a nucleic acid sequenceencoding a polypeptide, and transcriptional and translational stopsignals. The various nucleic acid and control sequences described abovemay be joined together to produce a recombinant expression vector whichmay include one or more convenient restriction sites to allow forinsertion or substitution of the promoter and/or nucleic acid sequenceencoding the polypeptide at such sites. Alternatively, the nucleic acidsequence may be expressed by inserting the nucleic acid sequence or anucleic acid construct comprising the promoter and/or sequence into anappropriate vector for expression. In creating the expression vector,the coding sequence is located in the vector so that the coding sequenceis operably linked with a promoter of the present invention and one ormore appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) which can be conveniently subjected to recombinant DNA proceduresand can bring about the expression of the nucleic acid sequence. Thechoice of the vector will typically depend on the compatibility of thevector with the host cell into which the vector is to be introduced. Thevectors may be linear or closed circular plasmids.

The vector may be an autonomously replicating vector, i.e., a vectorwhich exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. Furthermore, asingle vector or plasmid or two or more vectors or plasmids whichtogether contain the total DNA to be introduced into the genome of thehost cell, or a transposon may be used.

The vectors of the present invention preferably contain one or moreselectable markers which permit easy selection of transformed cells. Aselectable marker is a gene the product of which provides for biocide orviral resistance, resistance to heavy metals, prototrophy to auxotrophs,and the like. Suitable markers for yeast host cells are ADE2, HIS3,LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in afilamentous fungal host cell include, but are not limited to, amdS(acetamidase), argB (ornithine carbamoyltransferase), bar(phosphinothricin acetyltransferase), hygB (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),trpC (anthranilate synthase), as well as equivalents thereof Preferredfor use in an Aspergillus cell are the amdS and pyrG genes ofAspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

The vectors of the present invention preferably contain an element(s)that permits stable integration of the vector into the host cell'sgenome or autonomous replication of the vector in the cell independentof the genome.

For integration into the host cell genome, the vector may rely on thenucleic acid sequence encoding the polypeptide or any other element ofthe vector for stable integration of the vector into the genome byhomologous or nonhomologous recombination. Alternatively, the vector maycontain additional nucleic acid sequences for directing integration byhomologous recombination into the genome of the host cell. Theadditional nucleic acid sequences enable the vector to be integratedinto the host cell genome at a precise location(s) in the chromosome(s).To increase the likelihood of integration at a precise location, theintegrational elements should preferably contain a sufficient number ofnucleic acids, such as 100 to 1,500 base pairs, preferably 400 to 1,500base pairs, and most preferably 800 to 1,500 base pairs, which arehighly homologous with the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding nucleic acid sequences. On the other hand, thevector may be integrated into the genome of the host cell bynon-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. Examples of origins of replication for use in a yeasthost cell are the 2 micron origin of replication, ARS 1, ARS4, thecombination of Ars 1 and CEN3, and the combination of ARS4 and CEN6. Theorigin of replication may be one having a mutation which makes itsfunctioning temperature-sensitive in the host cell (see, e.g., Ehrlich,1978, Proceedings of the National Academy of Sciences USA 75: 1433).

More than one copy of a nucleic acid sequence encoding a polypeptide maybe inserted into the host cell to increase production of the geneproduct. An increase in the copy number of the nucleic acid sequence canbe obtained by integrating at least one additional copy of the sequenceinto the host cell genome or by including an amplifiable selectablemarker gene with the nucleic acid sequence where cells containingamplified copies of the selectable marker gene, and thereby additionalcopies of the nucleic acid sequence, can be selected for by cultivatingthe cells in the presence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga promoter of the present invention operably linked to a nucleic acidsequence encoding a polypeptide, which are advantageously used in therecombinant production of the polypeptides. A vector comprising apromoter of the present invention operably linked to a nucleic acidsequence encoding a polypeptide is introduced into a host cell so thatthe vector is maintained as a chromosomal integrant or as aself-replicating extra-chromosomal vector as described earlier. The term“host cell” encompasses any progeny of a parent cell that is notidentical to the parent cell due to mutations that occur duringreplication. The choice of a host cell will to a large extent dependupon the gene encoding the polypeptide and its source.

The host cell may be any fungal cell usefull in the methods of thepresent invention. “Fungi” as used herein includes the phyla Ascomycota,Basidiomycota, Chytridiomycota, and Zygomycota (as defined by Hawksworthet al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,1995, CAB International, University Press, Cambridge, UK) as well as theOomycota (as cited in Hawksworth et al., 1995, supra, page 171) and allmitosporic fungi (Hawksworth et al., 1995, supra).

In a preferred embodiment, the fungal host cell is a yeast cell. “Yeast”as used herein includes ascosporogenous yeast (Endomycetales),basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti(Blastomycetes). Since the classification of yeast may change in thefuture, for the purposes of this invention, yeast shall be defined asdescribed in Biology and Activities of Yeast (Skinner, F. A., Passmore,S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium SeriesNo. 9, 1980).

In a more preferred embodiment, the yeast host cell is a Candida,Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, orYarrowia cell.

In a most preferred embodiment, the yeast host cell is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensisor Saccharomyces oviformis cell. In another most preferred embodiment,the yeast host cell is a Kluyveromyces lactis cell. In another mostpreferred embodiment, the yeast host cell is a Yarrowia lipolytica cell.

In another preferred embodiment, the fungal host cell is a filamentousfungal cell. “Filamentous fungi” include all filamentous forms of thesubdivision Eumycota and Oomycota (as defined by Hawksworth et al, 1995,supra). The filamentous fungi are characterized by a mycelial wallcomposed of chitin, cellulose, glucan, chitosan, mannan, and othercomplex polysaccharides. Vegetative growth is by hyphal elongation andcarbon catabolism is obligately aerobic. In contrast, vegetative growthby yeasts such as Saccharomyces cerevisiae is by budding of aunicellular thallus and carbon catabolism may be fermentative.

In a more preferred embodiment, the filamentous fungal host cell is acell of a species of, but not limited to, Acremonium, Aspergillus,Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium,Thielavia, Tolypocladium, or Trichoderma.

In a most preferred embodiment, the filamentous fungal host cell is anAspergillus awamori, Aspergillus foetidus, Aspergillus japonicus,Aspergillus nidulans, Aspergillus niger or Aspergillus oryzae cell. Inanother most preferred embodiment, the filamentous fungal host cell is aFusarium bactridioides, Fusarium cerealis, Fusarium crookwellense,Fusarium culmorum, Fusarium graminearun, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusariun roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum,Fusarium trichothecioides, or Fusarium venenatum cell. In another mostpreferred embodiment, the filamentous fungal host cell is a Humicolainsolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila,Neurospora crassa, Penicillium purpurogenum, Thielavia terrestris,Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

In an even most preferred embodiment, the Fusarium venenatum cell isFusarium venenatum A3/5, which was originally deposited as Fusariumgraminearum ATCC 20334 and recently reclassified as Fusarium venenatumby Yoder and Christianson, 1998, Fungal Genetics and Biology 23: 62-80and O'Donnell et al., 1998, Fungal Genetics and Biology 23: 57-67; aswell as taxonomic equivalents of Fusarium venenatum regardless of thespecies name by which they are currently known. In another preferredembodiment, the Fusarium venenatum cell is a morphological mutant ofFusarium venenatum A3/5 or Fusarium venenatum ATCC 20334, as disclosedin WO 97/26330.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus host cells are described in EP 238 023 andYelton et al., 1984, Proceedings of the National Academy of Sciences USA81: 1470-1474. Suitable methods for transforming Fusarium species aredescribed by Malardier et al., 1989, Gene 78: 147-156 and WO 96/00787.Yeast may be transformed using the procedures described by Becker andGuarente, In Abelson, J. N. and Simon, M. I., editors, Guide to YeastGenetics and Molecular Biology, Methods in Enzymology, Volume 194, pp182-187, Academic Press, Inc., New York; Ito et al., 1983, Journal ofBacteriology 153: 163; and Hinnen et al., 1978, Proceedings of theNational Academy of Sciences USA 75: 1920.

Methods For Obtaining Mutant Promoters

The present invention further relates to methods for obtaining a mutantpromoter, comprising (a) introducing at least one mutation into thesequence of nucleotides 1 to 3949 of SEQ ID NO:1, nucleotides 1 to 938of SEQ ID NO:2, or nucleotides 1 to 3060 of SEQ ID NO:3, wherein themutant promoter has promoter activity; and (b) isolating the mutantpromoter.

The present invention also relates to methods for obtaining a mutantpromoter by (a) hybridizing a DNA under very low, low, medium,medium-high, high, or very high stringency conditions with (i) SEQ IDNO:1, SEQ ID NO:2, or SEQ ID NO:3, (ii) the cDNA sequence contained inSEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, (iii) a subsequence of (i) or(ii), or (iv) a complementary strand of (i), (ii), or (iii); and (b)isolating the mutant promoter from the DNA. Stringency and washconditions are defined herein.

The mutant promoter may be isolated using methods known in the art suchas restriction enzyme digestion or PCR amplification.

Nucleic Acid Sequences

The present invention also relates to isolated nucleic acid sequences,selected from the group consisting of:

(a) a nucleic acid sequence encoding a polypeptide having an amino acidsequence which has at least 65% identity with amino acids 22 to 581 ofSEQ ID NO:4, at least 65% identity with amino acids 19 to 200 of SEQ IDNO:5, or at least 65% identity with amino acids 1 to 187 of SEQ ID NO:6;

(b) a nucleic acid sequence having at least 65% homology withnucleotides 4013 to 5743 of SEQ ID NO:1, at least 65% homology withnucleotides 993 to 1593 of SEQ ID NO:2, or at least 65% homology withnucleotides 3061 to 3678 of SEQ ID NO:3;

(c) a nucleic acid sequence which hybridizes under very low, low,medium, medium-high, high, or very high stringency conditions with (i)nucleotides 4013 to 5743 of SEQ ID NO:1, nucleotides 993 to 1593 of SEQID NO:2, or nucleotides 3061 to 3678 of SEQ ID NO:3; (ii) the cDNAsequence contained in nucleotides 4013 to 5743 of SEQ ID NO:1,nucleotides 993 to 1593 of SEQ ID NO:2, or nucleotides 3061 to 3678 ofSEQ ID NO:3; (iii) a subsequence of (i) or (ii) of at least 100nucleotides, or (iv) a complementary strand of (i), (ii), or (iii);

(d) a nucleic acid sequence encoding a variant of the polypeptide havingan amino acid sequence of SEQ ID NO:4, SEQ ID. NO. 5, or SEQ ID NO:6comprising a substitution, deletion, and/or insertion of one or moreamino acids;

(e) an allelic variant of (a), (b), or (c); and

(f) a subsequence of (a), (b), (c), or (e).

The term “isolated nucleic acid sequence” as used herein refers to anucleic acid sequence which is essentially free of other nucleic acidsequences, e.g., at least about 20% pure, preferably at least about 40%pure, more preferably at least about 60% pure, even more preferably atleast about 80% pure, and most preferably at least about 90% pure asdetermined by agarose electrophoresis. For example, an isolated nucleicacid sequence can be obtained by standard cloning procedures used ingenetic engineering to relocate the nucleic acid sequence from itsnatural location to a different site where it will be reproduced. Thecloning procedures may involve excision and isolation of a desirednucleic acid fragment comprising the nucleic acid sequence encoding thepolypeptide, insertion of the fragment into a vector molecule, andincorporation of the recombinant vector into a host cell where multiplecopies or clones of the nucleic acid sequence will be replicated. Thenucleic acid sequence may be of genomic, cDNA, RNA, semisynthetic,synthetic origin, or any combinations thereof.

In a first embodiment, the present invention relates to isolated nucleicacid sequences encoding polypeptides having an amino acid sequence whichhas a degree of identity to amino acids 22 to 581 of SEQ ID NO:4, aminoacids 19 to 200 of SEQ ID NO:5, or amino acids 1 to 187 of SEQ ID NO:6(i.e., the mature polypeptide) of at least about 65%, preferably atleast about 70%, more preferably at least about 80%, even morepreferably at least about 90%, most preferably at least about 95%, andeven most preferably at least about 97% (hereinafter “homologouspolypeptides”). In a preferred embodiment, the homologous polypeptideshave an amino acid sequence which differs by five amino acids,preferably by four amino acids, more preferably by three amino acids,even more preferably by two amino acids, and most preferably by oneamino acid from amino acids 22 to 581 of SEQ ID NO:4, amino acids 19 to200 of SEQ ID NO:5, or amino acids 1 to 187 of SEQ ID NO:6. For purposesof the present invention, the degree of identity between two amino acidsequences is determined by the Clustal method (Higgins, 1989, CABIOS 5:151-153) using the LASERGENE™ MEGALIGN™ software (DNASTAR, Inc.,Madison, Wis.) with an identity table and the following multiplealignment parameters: Gap penalty of 10, and gap length penalty of 10.Pairwise alignment parameters were Ktuple=1, gap penalty=3, windows=5,and diagonals=5.

Preferably, the nucleic acid sequences of the present invention encodepolypeptides that comprise the amino acid sequence of SEQ ID NO:4, SEQID NO:5, or SEQ ID NO:6; or an allelic variant thereof; or a fragmentthereof. In a more preferred embodiment, the nucleic acid sequence ofthe present invention encodes a polypeptide that comprises the aminoacid sequence of SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6. In anotherpreferred embodiment, the nucleic acid sequence of the present inventionencodes a polypeptide that comprises amino acids 22 to 581 of SEQ IDNO:4, amino acids 19 to 200 of SEQ ID NO:5, or amino acids 1 to 187 ofSEQ ID NO:6; or an allelic variant thereof; or a fragment thereof. Inanother preferred embodiment, the nucleic acid sequence of the presentinvention encodes a polypeptide that comprises amino acids 22 to 581 ofSEQ ID NO:4, amino acids 19 to 200 of SEQ ID NO:5, or amino acids 1 to187 of SEQ ID NO:6. In another preferred embodiment, the nucleic acidsequence of the present invention encodes a polypeptide that consists ofthe amino acid sequence of SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6; oran allelic variant thereof; or a fragment thereof. In another preferredembodiment, the nucleic acid sequence of the present invention encodes apolypeptide that consists of the amino acid sequence of SEQ ID NO:4, SEQID NO:5, or SEQ ID NO:6. In another preferred embodiment, the nucleicacid sequence of the present invention encodes a polypeptide thatconsists of amino acids 22 to 581 of SEQ ID NO:4, amino acids 19 to 200of SEQ ID NO:5, or amino acids 1 to 187 of SEQ ID NO:6; or an allelicvariant thereof; or a fragment thereof. In another preferred embodiment,the nucleic acid sequence of the present invention encodes a polypeptidethat consists of amino acids 22 to 581 of SEQ ID NO:4, amino acids 19 to200 of SEQ ID NO:5, or amino acids 1 to 187 of SEQ ID NO:6.

The present invention also encompasses nucleic acid sequences whichencode a polypeptide having the amino acid sequence of SEQ ID NO:4, SEQID NO:5, or SEQ ID NO:6, which differ from SEQ ID NO:1, SEQ ID NO:2, orSEQ ID NO:3, respectively, by virtue of the degeneracy of the geneticcode. The present invention also relates to subsequences of SEQ ID NO:1,SEQ ID NO:2, and SEQ ID NO:3 which encode fragments of SEQ ID NO:4, SEQID NO:5, and SEQ ID NO:6, respectively.

A subsequence is a nucleic acid sequence encompassed by SEQ ID NO:1, SEQID NO:2, or SEQ ID NO:3, except that one or more nucleotides from the 5′and/or 3′ end have been deleted. Preferably, a subsequence of SEQ IDNO:1contains at least 1410 nucleotides, more preferably at least 1500nucleotides, and most preferably at least 1590 nucleotides. Preferably,a subsequence of SEQ ID NO:2 contains at least 450 nucleotides, morepreferably at least 480 nucleotides, and most preferably at least 510nucleotides. Preferably, a subsequence of SEQ ID NO:3 contains at least465 nucleotides, more preferably at least 495 nucleotides, and mostpreferably at least 525 nucleotides. A fragment of SEQ ID NO:4, SEQ IDNO:6, or SEQ ID NO:6 is a polypeptide having one or more amino acidsdeleted from the amino and/or carboxy terminus of this amino acidsequence. Preferably, a fragment of SEQ ID NO:4 contains at least 470amino acid residues, more preferably at least 500 amino acid residues,and most preferably at least 530 amino acid residues. Preferably, afragment of SEQ ID NO:5 contains at least 150 amino acid residues, morepreferably at least 160 amino acid residues, and most preferably atleast 170 amino acid residues. Preferably, a fragment of SEQ ID NO:6contains at least 155 amino acid residues, more preferably at least 165amino acid residues, and most preferably at least 175 amino acidresidues.

An allelic variant denotes any of two or more alternative forms of agene occupying the same chomosomal locus. Allelic variation arisesnaturally through mutation, and may result in polymorphism withinpopulations. Gene mutations can be silent (no change in the encodedpolypeptide) or may encode polypeptides having altered amino acidsequences. The allelic variant of a polypeptide is a polypeptide encodedby an allelic variant of a gene.

In a second embodiment, the present invention relates to isolatednucleic acid sequences which have a degree of homology to nucleotides4013 to 5743 of SEQ ID NO:1, nucleotides 4013 to 1593 of SEQ ID NO:2, ornucleotides 3061 to 3678 of SEQ ID NO:3 (i.e., mature polypeptide codingregion) of at least about 65%, preferably about 70%, preferably about80%, more preferably about 90%, even more preferably about 95%, and mostpreferably about 97% homology, which encode an active polypeptide; orallelic variants and subsequences thereof. For purposes of the presentinvention, the degree of homology between two nucleic acid sequences isdetermined by the Wilbur-Lipman method (Wilbur and Lipman, 1983,Proceedings of the National Academy of Science USA 80: 726-730) usingthe LASERGENE™ MEGALIGN™ software (DNASTAR, Inc., Madison, Wis.) with anidentity table and the following multiple alignment parameters: Gappenalty of 10 and gap length penalty of 10. Pairwise alignmentparameters were Ktuple=3, gap penalty=3, and windows=20.

In a third embodiment, the present invention relates to isolated nucleicacid sequences encoding polypeptides which hybridize under very lowstringency conditions, preferably low stringency conditions, morepreferably medium stringency conditions, more preferably medium-highstringency conditions, even more preferably high stringency conditions,and most preferably very high stringency conditions with a nucleic acidprobe which hybridizes under the same conditions with (i) nucleotides4013 to 5743 of SEQ ID NO:1, nucleotides 993 to 1593 of SEQ ID NO:2, ornucleotides 3061 to 3678 of SEQ ID NO:3; (ii) the cDNA sequencecontained in nucleotides 4013 to 5743 of SEQ ID NO:1, nucleotides 993 to1593 of SEQ ID NO:2, or nucleotides 3061 to 3678 of SEQ ID NO:3; (iii) asubsequence of (i) or (ii), or (iv) a complementary strand of (i), (ii),or (iii) (J. Sambrook, E. F. Fritsch, and T. Maniatus, 1989, MolecularCloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, N.Y.). Thesubsequence of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3 may be at least100 nucleotides or preferably at least 200 nucleotides. Moreover, thesubsequence may encode a polypeptide fragment.

The nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3,or a subsequence thereof, as well as the amino acid sequence of SEQ IDNO:4, SEQ ID NO:5, or SEQ ID NO:6, or a fragment thereof, may be used todesign a nucleic acid probe to identify and clone DNA encodingpolypeptides from strains of different genera or species according tomethods well known in the art. In particular, such probes can be usedfor hybridization with the genomic or cDNA of the genus or species ofinterest, following standard Southern blotting procedures, in order toidentify and isolate the corresponding gene therein. Such probes can beconsiderably shorter than the entire sequence, but should be at least15, preferably at least 25, and more preferably at least 35 nucleotidesin length. Longer probes can also be used. Both DNA and RNA probes canbe used. The probes are typically labeled for detecting thecorresponding gene (for example, with ³²P, ³H, ³⁵S, biotin, or avidin).Such probes are encompassed by the present invention.

Thus, a genomic DNA or cDNA library prepared from such other organismsmay be screened for DNA which hybridizes with the probes described aboveand which encodes a polypeptide. Genomic or other DNA from such otherorganisms may be separated by agarose or polyacrylamide gelelectrophoresis, or other separation techniques. DNA from the librariesor the separated DNA may be transferred to and immobilized onnitrocellulose or other suitable carrier material. In order to identifya clone or DNA which is homologous with SEQ ID NO:1 or a subsequencethereof, the carrier material is used in a Southern blot. For purposesof the present invention, hybridization indicates that the nucleic acidsequence hybridizes to a labeled nucleic acid probe corresponding to thenucleic acid sequence shown in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3,their complementary strands, or subsequences thereof, under very low tovery high stringency conditions. Molecules to which the nucleic acidprobe hybridizes under these conditions are detected using X-ray film.

In a preferred embodiment, the nucleic acid probe is the nucleic acidsequence of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. In anotherpreferred embodiment, the nucleic acid probe is a nucleic acid sequencewhich encodes the polypeptide of SEQ ID NO:4, SEQ ID NO:5, or SEQ IDNO:6, or subsequences thereof. In another preferred embodiment, thenucleic acid probe is SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. Inanother preferred embodiment, the nucleic acid probe is nucleotides 4013to 5743 of SEQ ID NO:1, which encodes a mature polypeptide. In anotherpreferred embodiment, the nucleic acid probe is nucleotides 993 to 5743of SEQ ID NO:2, which encodes a mature polypeptide. In another preferredembodiment, the nucleic acid probe is nucleotides 3061 to 3678 of SEQ IDNO:3, which encodes a mature polypeptide. In another preferredembodiment, the nucleic acid probe is the nucleic acid sequencecontained in plasmid pECO3 which is contained in Escherichia coli NRRLB-30067. In another preferred embodiment, the nucleic acid probe is thenucleic acid sequence contained in plasmid pFAMG which is contained inEscherichia coli NRRL B-30071. In another preferred embodiment, thenucleic acid probe is the nucleic acid sequence contained in plasmidpQUINN which is contained in Escherichia coli NRRL B-30076. In anotherpreferred embodiment, the nucleic acid probe is the mature polypeptidecoding region contained in plasmid pECO3 which is contained inEscherichia coli NRRL B-30067. In another preferred embodiment, thenucleic acid probe is the mature polypeptide coding region contained inplasmid pFAMG which is contained in Escherichia coli NRRL B-30071. Inanother preferred embodiment, the nucleic acid probe is the maturepolypeptide coding region contained in plasmid pQUINN which is containedin Escherichia coli NRRL B-30076.

For long probes of at least 100 nucleotides in length, very low to veryhigh stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared anddenatured salmon sperm DNA, and either 25% formamide for very low andlow stringencies, 35% formamide for medium and medium-high stringencies,or 50% formamide for high and very high stringencies, following standardSouthern blotting procedures.

For long probes of at least 100 nucleotides in length, the carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS preferably at least at 45° C. (very low stringency), morepreferably at least at 50° C. (low stringency), more preferably at leastat 55° C. (medium stringency), more preferably at least at 60° C.(medium-high stringency), even more preferably at least at 65° C. (highstringency), and most preferably at least at 70° C. (very highstringency).

For short probes which are about 15 nucleotides to about 70 nucleotidesin length, stringency conditions are defined as prehybridization,hybridization, and washing post-hybridization at 5° C. to 10° C. belowthe calculated T_(m) using the calculation according to Bolton andMcCarthy (1962, Proceedings of the National Academy of Sciences USA48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA, 0.5% NP40,1×Denhardt's solution, 1 mM sodium pyrophosphate, 1 mM sodium monobasicphosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per ml following standardSouthern blotting procedures.

For short probes which are about 15 nucleotides to about 70 nucleotidesin length, the carrier material is washed once in 6×SCC plus 0.1% SDSfor 15 minutes and twice each for 15 minutes using 6×SSC at 5° C. to 10°C. below the calculated T_(m).

The present invention also relates to isolated nucleic acid sequencesproduced by (a) hybridizing a DNA under very low, low, medium,medium-high, high, or very high stringency conditions with (i)nucleotides 4013 to 5743 of SEQ ID NO:1, nucleotides 993 to 1593 of SEQID NO:2, or nucleotides 3061 to 3678 of SEQ ID NO:3, (ii) the cDNAsequence contained in nucleotides 4013 to 5743 of SEQ ID NO:1,nucleotides 993 to 1593 of SEQ ID NO:2, nucleotides 3061 to 3678 of SEQID NO:3, (iii) a subsequence of (i) or (ii), or (iv) a complementarystrand of (i), (ii), or (iii); and (b) isolating the nucleic acidsequence from the DNA. The subsequence is preferably a sequence of atleast 100 nucleotides such as a sequence which encodes a polypeptidefragment.

In a fourth embodiment, the present invention relates to isolatednucleic acid sequences which encode variants of the polypeptide havingan amino acid sequence of SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6comprising a substitution, deletion, and/or insertion of one or moreamino acids.

The amino acid sequences of the variant polypeptides may differ from theamino acid sequence of SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6, or themature polypeptides thereof, by an insertion or deletion of one or moreamino acid residues and/or the substitution of one or more amino acidresidues by different amino acid residues. Preferably, amino acidchanges are of a minor nature, that is conservative amino acidsubstitutions that do not significantly affect the folding and/oractivity of the protein; small deletions, typically of one to about 30amino acids; small amino- or carboxyl-terminal extensions, such as anamino-terminal methionine residue; a small linker peptide of up to about20-25 residues; or a small extension that facilitates purification bychanging net charge or another function, such as a poly-bistidine tract,an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the group of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions which do not generally alter the specific activityare known in the art and are described, for example, by H. Neurath andR. L. Hill, 1979, In, The Proteins, Academic Press, New York. The mostcommonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser,Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg,Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly as well as these inreverse.

The nucleic acid sequences of the present invention may be obtained frommicroorganisms of any genus. For purposes of the present invention, theterm “obtained from” is used as defined herein. In a preferredembodiment, the polypeptide encoded by a nucleic acid sequence of thepresent invention is secreted extracellularly.

The nucleic acid sequences may be obtained from a fungal source, andmore preferably from a yeast strain such as a Candida, Hansenula,Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowiastrain; or more preferably from a filamentous fungal strain such as anAcremonium, Aspergillus, Aureobasidium, Cryptococcus, Filibasidium,Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix,Neurospora, Paecilomyces, Penicillium, Pironzyces, Schizophyllum,Talaroinyces, Thermoascus, Thielavia, Tolypocladium, or Trichodermastrain.

In a preferred embodiment, the nucleic acid sequences are obtained froma Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomycesdiastaticus, Saccharomyces douglasii, Saccharomyces kluyveri,Saccharomyces norbensis or Saccharomyces oviformis strain.

In another preferred embodiment, the nucleic acid sequences are obtainedfrom an Aspergillus aculeatus, Aspergillus awamori, Aspergillusfoetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillusniger, Aspergillus oryzae, Humicola insolens, Humicola lanuginosa, Mucormiehei, Myceliophthora thermophila, Neurospora crassa, Penicilliumpurpurogenum, Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, or Trichoderma viride strain.

In another preferred embodiment, the nucleic acid sequences are obtainedfrom a Fusarium bactridioides, Fusarium cerealis, Fusariumcrookwellense, Fusarium culmorum, Fusarium graminearum, Fusariumgraminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum,Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusariumsarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusariumtorulosum, Fusarium trichothecioides, Fusarium venenatum strain.

In a more preferred embodiment, the nucleic acid sequences are obtainedfrom Fusarium venenatum, and most preferably from Fusariuin venenatumATCC 20334, e.g., the nucleic acid sequence set forth in SEQ ID NO:1,SEQ ID NO:2, or SEQ ID NO:3. In another more preferred embodiment, thenucleic acid sequence of SEQ ID NO:1 is the sequence contained inplasmid pECO3 which is contained in Escherichia coli NRRL B-30067. Inanother more preferred embodiment, the nucleic acid sequence of SEQ IDNO:2 is the sequence contained in plasmid pFAMG which is contained inEscherichia coli NRRL B-30071. In another more preferred embodiment, thenucleic acid sequence of SEQ ID NO:3 is the sequence contained inplasmid pQUINN which is contained in Escherichia coli NRRL B-300076. Inanother preferred embodiment, the nucleic acid sequence is nucleotides4013 to 5743 of SEQ ID NO: 1, which encodes a mature polypeptide. Inanother preferred embodiment, the nucleic acid sequence is nucleotides993 to 1593 of SEQ ID NO:2, which encodes a mature polypeptide. Inanother preferred embodiment, the nucleic acid sequence is nucleotides3061 to 3678 of SEQ ID NO:3, which encodes a mature polypeptide.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), andAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

Furthermore, such nucleic acid sequences may be identified and obtainedfrom other sources including microorganisms isolated from nature (e.g.,soil, composts, water, etc.) using the above-mentioned probes.Techniques for isolating microorganisms from natural habitats are wellknown in the art. The nucleic acid sequence may then be derived bysimilarly screening a genomic or cDNA library of another microorganism.Once a nucleic acid sequence encoding a polypeptide has been detectedwith the probe(s), the sequence may be isolated or cloned by utilizingtechniques which are known to those of ordinary skill in the art (see,e.g., Sambrook et al., 1989, supra).

The present invention also relates to mutant nucleic acid sequencescomprising at least one mutation in the mature polypeptide codingsequence of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, in which themutant nucleic acid sequence encodes a polypeptide which consists ofamino acids 22 to 581 of SEQ ID NO:4, amino acids 19 to 200 of SEQ IDNO:5, or amino acids 1 to 187 of SEQ ID NO:6, respectively.

The techniques used to isolate or clone a nucleic acid sequence encodinga polypeptide have been described herein.

The present invention also relates to nucleic acid constructs,recombinant vectors, and host cells containing the nucleic acidsequences described above. The same methods may be used as describedearlier for their construction.

The present invention further relates to the polypeptides encoded by thenucleic acid sequences described above as well as methods of productionof the polypeptides using the methods described herein.

The present invention is further described by the following exampleswhich should not be construed as limiting the scope of the invention.

EXAMPLES

Chemicals used as buffers and substrates were commercial products of atleast reagent grade.

Media and Solutions

COVE trace metals solution was composed per liter of 0.04 g of NaB₄O₇.10H₂O, 0.4 g of CuSO₄. 5H₂O, 1.2 g of FeSO₄. 7H₂O, 0.7 g of MnSO₄. H₂O,0.8 g of Na₂MoO₂. 2H₂O, and 10 g of ZnSO₄. 7H₂O.

50×COVE salts solution was composed per liter of 26 g of KCl, 26 g ofMgSO₄. 7H₂O, 76 g of KH₂PO₄, and 50 ml of COVE trace metals.

COVE medium was composed per liter of 342.3 g of sucrose, 20 ml of50×COVE salt solution, 10 ml of 1 M acetamide, 10 ml of 1.5 M CsCl₂, and25 g of Noble agar.

50×Vogels medium was composed per liter of 150 g of sodium citrate, 250g of KH₂PO₄, 10 g of MgSO₄. 7H₂O, 10 g of CaCl₂. 2H₂O, 2.5 ml of biotinstock solution, and 5.0 ml of AMG trace metals solution.

? Trace metals solution was composed per liter of 14.3 g of ZnSO₄. 7H₂O,2.5 g of CuSO₄. 5H₂O, 0.5 g of NiCl₂, 13:8 g of FeSO₄, 8.5 g of MnSO₄,and 3.0 g of citric acid.

COVE top agarose was composed per liter of 20 ml of 50×COVE salts, 0.8 Msucrose, 1.5 M cesium chloride, 1.0 M acetamide, and 10 g of low meltagarose, pH adjusted to 6.0.

BASTA top agarose was composed of COVE top agarose supplemented with 10mg/ml of the herbicide Basta™ (Hoechst Schering, Rodovre, Denmark).

RA sporulation medium was composed per liter of 50 g of succinic acid,12.1 g of NaNO₃, 1 g of glucose, 20 ml of 50×Vogels, and 0.5 ml of a 10mg/ml NaMoO₄ stock solution, pH to 6.0.

YEPG medium was composed per liter of 10 g of yeast extract, 20 g ofpeptone, and 20 g of glucose.

STC was composed of 0.8 M sorbitol, 25 mM Tris pH 8, 25 mM CaCl₂.

SPTC was composed of 40% PEG 4000, 0.8 M sorbitol, 25 mM Tris pH 8, 25mM CaCl₂.

M400Da medium was composed per liter of 50 g of maltodextrin, 2 g ofMgSO₄. 7H₂O, 2 g of KH₂PO₄, 4 g of citric acid, 8 g of yeast extract, 2g of urea, and 1 ml of COVE trace metals solution.

Example 1 Production of Fusarium venenatum Mycelial Tissue

Fusarium venenatum CCl-3, a morphological mutant of Fusarium strain ATCC20334 (Wiebe et al., 1991, Mycological Research 95: 1284-1288), wasgrown in a two-liter lab-scale fermentor using a fed-batch fermentationscheme with NUTRIOSE™ (Roquette Freres, S.A., Beinheim, France) as thecarbon source and yeast extract. Ammonium phosphate was provided in thefeed. The pH was maintained at 6 to 6.5, and the temperature was kept at30° C. with positive dissolved oxygen.

Mycelial samples were harvested at 2, 4, 6, and 8 days post-inoculum andquick-frozen in liquid nitrogen. The samples were stored at −80° C.until they were disrupted for RNA extraction.

Example 2 cDNA Library Construction

Total cellular RNA was extracted from the mycelial samples described inExample 1 according to the method of Timberlake and Barnard (1981, Cell26: 29-37), and the RNA samples were analyzed by Northern hybridizationafter blotting from 1% formaldehyde-agarose gels (Davis et al., 1986,Basic Methods in Molecular Biology, Elsevier Science Publishing Co.,Inc., New York). Polyadenylated mRNA fractions were isolated from totalRNA with an mRNA Separator Kit™ (Clontech Laboratories, Inc., Palo Alto,Calif.) according to the manufacturer's instructions. Double-strandedcDNA was synthesized using approximately 5 μg of poly(A)+mRNA accordingto the method of Gubler and Hoffman (1983, Gene 25: 263-269) except aNotI-(dT)18 primer (Pharmacia Biotech, Inc., Piscataway, N.J.) was usedto initiate first strand synthesis. The cDNA was treated with mung beannuclease (Boehringer Mannheim Corporation, Indianapolis, Ind.) and theends were made blunt with T4 DNA polymerase (New England Biolabs,Beverly, Mass.).

The cDNA was digested with NotI, size selected by agarose gelelectrophoresis (ca. 0.7-4.5 kb), and ligated with pZErO-2.1(Invitrogen, Carlsbad, Calif.) which had been cleaved with NotI plusEcoRV and dephosphorylated with calf-intestine alkaline phosphatase(Boehringer Mannheim Corporation, Indianapolis, Ind.). The ligationmixture was used to transform competent E. coli TOPIO cells (Invitrogen,Carlsbad, Calif.). Transformants were selected on 2YT agar plates(Miller, 1992, A Short Course in Bacterial Genetics. A Laboratory Manualand Handbook for Escherichia coli and Related Bacteria, Cold SpringHarbor Press, Cold Spring Harbor, N.Y.) which contained kanamycin at afinal concentration of 50 μg/ml.

Two independent directional cDNA libraries were constructed using theplasmid cloning vector pZErO-2.1. Library A was made using mRNA frommycelia harvested at four days, and Library B was constructed with mRNAfrom the six day time point. Neither cDNA library was amplified in orderto examine a representative “snapshot” of the gene expression profile inthe cells. Instead the libraries were plated, titered, and independentclones from each were analyzed by DNA sequencing.

Library A (4 day cells) consisted of about 7.5×10⁴ independent clonesand Library B (6 day cells) consisted of roughly 1.2×10⁵ clones.Miniprep DNA was isolated from forty colonies in each library andchecked for the presence and size of cDNA inserts. In this analysis 39of 40 colonies (97.5%) from Library A contained inserts with sizesranging from 600 bp to 2200 bp (avg.=1050 bp). Similarly, 39 of 40colonies (97.5%) from Library B had inserts with sizes ranging from 800bp to 3600 bp (avg.=1380 bp).

Example 3 Template Preparation and Nucleotide Sequencing

From each cDNA library described in Example 2, 1192 transformantcolonies were picked directly from the transformation plates into96-well microtiter dishes which contained 200 μl of 2YT broth (Miller,1992, supra) with 50 μg/ml kanamycin. The plates were incubatedovernight at 37° C. without shaking. After incubation 100 μl of sterile50% glycerol were added to each well. The transformants were replicatedinto secondary, deep-dish 96-well microculture plates (Advanced GeneticTechnologies Corporation, Gaithersburg, Md.) containing 1 ml ofMagnificent Broth™ (MacConnell Research, San Diego, Calif.) supplementedwith 50 μg of kanamycin per ml in each well. The primary microtiterplates were stored frozen at −80° C. The secondary deep-dish plates wereincubated at 37° C. overnight with vigorous agitation (300 rpm) onrotary shaker. To prevent spilling and cross-contamination, and to allowsufficient aeration, each secondary culture plate was covered with apolypropylene pad (Advanced Genetic Technologies Corporation,Gaithersburg, Md.) and a plastic microtiter dish cover.

DNA was isolated from each well using the 96-well Miniprep Kit protocolof Advanced Genetic Technologies Corporation (Gaithersburg, Md.) asmodified by Utterback et al. (1995, Genome Sci. Technol. 1: 1-8).Single-pass DNA sequencing was done with a Perkin-Elmer AppliedBiosystems Model 377 XL Automatic DNA Sequencer (Perkin-Elmer AppliedBiosystems, Inc., Foster City, Calif.) using dye-terminator chemistry(Giesecke et al., 1992, Journal of Virology Methods 38: 47-60) and thereverse lac sequencing primer.

Example 4 Analysis of DNA Sequence Data

Nucleotide sequence data were scrutinized for quality, and samplesgiving improper spacing less than or equal to 9.2 or ambiguity levelsexceeding 3% were discarded or re-run. Vector sequences were trimmedwith assistance of FACTURA™ software (Perkin-Elmer Applied Biosystems,Inc., Foster City, Calif.). In addition, sequences were truncated at theend of each sample when the number of ambiguous base calls increased.All sequences were compared to each other to construct overlappingcontigs using TIGR Assembler software (Sutton, G. G. et al., 1995,Genome Science and Technology 1: 9019) to determine multiplicity ofvarious cDNA species represented in each library. Lastly, all sequenceswere translated in three frames and searched against a non-redundantdata base (NRDB) using GeneAssist™ software (Perkin-Elmer AppliedBiosystems, Inc., Foster City, Calif.) with a modified Smith-Watermanalgorithm using the BLOSUM 62 matrix with a threshold score of 70. TheNRDB was assembled from Genpept, Swiss-Prot, and PIR databases.

Example 5 Identification of Glucoamylase cDNA Clones

Putative glucoamylase clones were identified by partial sequencing ofrandom cDNA clones using an Applied Biosystems Model 377 XL AutomatedDNA Sequencer according to the manufacturer's instructions andcomparison of the deduced amino acid sequence to the sequences in theNRDB as described in Example 4. From more than 2000 cDNA sequencesanalyzed, two clones from Library A and nine clones from Library Bshowed amino acid sequence homology to glucoamylase proteins from otherfungi and yeasts. Among several glucoamylase cDNA clones discovered inthis manner, one was estimated to be full-length (encoding the completeprotein) on the basis of its alignment to the Neurospora crassa (Geneseqprotein accession number R71034) and Humicola grisea (Trembi accessionnumber Q12623) glucoamylase amino acid sequences and the presence of apossible signal peptide, detected using the Signal-P computer program(Nielsen, et al., 1997, Protein Engineering 10: 1-6). This clonedesignated E. coli FA0401 containing plasmid pFA0401 was selected foruse as a probe to clone the corresponding glucoamylase genomic DNAsequence from Fusarium venenatum (see Example 7).

Example 6 Construction a Library of Fusarium venenatum Genomic DNA

Genomic libraries were constructed in λZipLox as specified by theprocedure of Berka et al. (1998, Appl. Environ. Microbiol. 64,4423-4427). Briefly, Fusarium venenatum total cellular DNA was partiallydigested with Tsp509I and size-fractionated on 1% agarose gels. DNAfragments migrating in the size range 3-7 kb were excised and elutedfrom the agarose gel slices using Prep-a-Gene reagents (BioRad,Hercules, Calif.). The eluted DNA fragments were ligated withEcoRI-cleaved and dephosphorylated λZipLox vector arms (LifeTechnologies, Gaithersburg, Md.), and the ligation mixtures werepackaged using commercial packaging extracts (Stratagene, La Jolla,Calif.). The packaged DNA libraries were plated and amplified onEscherichia coli Y1090ZL cells and stored at 4° C. using standardmethods (Davis, R. W., Botstein, D., and Roth, J. R., 1980, AdvancedBacterial Genetics, A Manual for Genetic Engineering, Cold Spring HarborPress, Cold Spring Harbor, N.Y.).

Example 7 Isolation, Nucleotide Sequencing and Characterization of aGenomic DNA Segment Encoding Fusarium venenatum Glucoamylase

Approximately 50,000 plaques from the Fusarium venenatum genomic DNAlibrary (Example 6) were screened by hybridization (Davis et al., 1980,supra) with a radiolabeled probe fragment comprising the cloned cDNAinsert from pFA0401 (Example 5) using high stringency conditions (i.e.,hybridization at 45° C. in 50% formamide, 5×SSPE, 0.3% SDS, 200 μg/mlsheared and denatured salmon sperm DNA; filters washed once in 0.2×SSPEwith 0.1% SDS at 45° C. followed by two washes in 0.2×SSPE with no SDSat the same temperature). Plaques which gave hybridization signals, werepurified twice on E. coli Y1090ZL cells, and the individual clones weresubsequently excised from the λZipLox vector as pZL1-derivatives(D'Alessio et al., 1992, Focus® 14: 76). DNA sequence analysis revealedthat one of these clones, containing a plasmid designated pFAMG,comprised the entire glucoamylase coding region as well as 3.9 kb of5′-flanking and 0.3 kb of 3′-flanking DNA encompassing the promoter andterminator regions, respectively (see FIG. 1).

DNA sequencing of the cloned insert in pFAMG was done with an AppliedBiosystems Model 377 XL Automated DNA Sequencer using dye-terminatorchemistry. Contiguous sequences were generated using a transposoninsertion strategy (Primer Island Transposition Kit,Perkin-Elmer/Applied Biosystems, Inc., Foster City, Calif.), and theglucoamylase genomic clone from pFAMG was sequenced to an averageredundancy of 4.2.

By comparing the genomic sequence data to the contig of glucoamylasecDNA sequences, it was determined that the genomic DNA segment encodingFusarium venenatum glucoamylase contained an open reading frame of 1743bp interrupted by an intron of 51 bp and encoding a polypeptide of 581amino acids. The nucleotide sequence (SEQ ID NO:1) and deduced aminoacid sequence (SEQ ID NO:4) are shown in FIG. 1. Using the SignalPprogram (Nielsen et al., 1997, Protein Engineering 10: 1-6), a signalpeptide of 21 residues was predicted, and confirmed by amino terminalsequencing of the glucoamylase protein (Example 8). Thus, the matureglucoamylase is composed of 560 amino acids.

A comparative alignment of fungal glucoamylase protein sequences wasundertaken using the Clustal method (Higgins, 1989, CABIOS 5: 151-153)using the LASERGENE™ MEGALIGN™ software (DNASTAR, Inc., Madison, Wis.)with an identity table and the following multiple alignment parameters:Gap penalty of 10 and gap length penalty of 10. Pairwise alignmentparameters were Ktuple=1, gap penalty=3, windows=5, and diagonals=5. Thealignment showed that the Fusarium venenatum glucoamylase sharesidentity with the following glucoamylases from other fungi (percentidentical residues in parentheses): Neurospora crassa [Swissprot P14804](47%), Aspergillus niger [Swissprot P04064] (46%), Humicola grisea[Trembl Q12623] (44%), Hormoconis resinae [Swissprot Q03045] (41%),Corticium rolfsii [Q12596] (41%), Schizosaccharomyces pombe [Trembl060087] (31%), and Rhizopus oryzae [Swissprot P07683] (23%).

Example 8 Amino Terminal Sequence Analysis of Fusarium venenatumGlucoamylase

The proteins in a sample of Fusarium venenatum fermentation broth(Example 1) were separated by 8-16% Tris-glycine SDS-PAGE (Novex, SanDiego, Calif.) and electroblotted onto a PVDF membrane (Novex, SanDiego, Calif.) using 10 mM CAPS (3-[cyclohexylamino]-l-propanesulfonicacid) in 10% methanol, pH=11.0 for 2 hours at 25 volts. The PVDFmembrane was stained with 0.1% Commassie Blue R-250 in 40% methanol/1%acetic acid for 20 seconds and destained in 50% ethanol to observe theprotein bands.

The major polypeptide species migrating at approximately 65 kDa wasexcised and subjected to N-terminal sequencing on an Applied Biosystems476A Protein Sequencer (Perkin Elmer/Applied Biosystems Division, FosterCity, Calif.) with on-line HPLC and liquid phase trifluoroacetic acid(TFA) delivery. Sequencing reagents were from Perkin Elmer/AppliedBiosystems Division. (Foster City, Calif.). Detection ofphenylthiohydantoin-amino acids was accomplished by on-line HPLC usingBuffer A containing 3.5% tetrahydrofuran in water with 18 ml of thePremix concentrate (Perkin Elmer/Applied Biosystems Division, FosterCity, Calif.) containing acetic acid, sodium acetate, and sodiumhexanesulfonate and Buffer B containing Acetonitrile. Data was collectedand analyzed on a Macintosh IIsi using Applied Biosystems 610 DataAnalysis Software. Amino acid identifications were performed byvisualizing chromatograms against a light source and determined by theoperator. The N-terminal analysis yielded a protein sequence ofSer-Pro-Ser-Lys-Asp-Asn-Ser-Leu-Glu-Arg-Phe-Ile-Asp-Lys-Gln-Ala-Asp-Ile-Ser(SEQ ID NO:4).

Example 9 Identification of Abundant cDNA Clones Encoding an PutativeVacuolar Associated Protein and an Unknown Secreted Gene Product

Two additional cDNA species were selected based on their relativeabundance among the clones present in Libraries A and B (Example 2).

Clone FA0035, containing pFA0035, encoded a primary translation productof 200 amino acids with a possible signal peptide of 18 amino acids thatwas detected using the Signal-P computer program (Nielsen, et al., 1997,supra). Clone FA0035 was represented by approximately 1.9% of the cDNAclones in Library A and 0.8% of Library B. Comparison of the deducedamino acid sequence of clone FA0035 to the sequences in the NRDB asdescribed in Example 4 revealed no significant homology to any knownprotein sequences, and it is therefore categorized as an unknown openreading frame (ORF). The name “Daria” was arbitrarily assigned to thisclone.

Clone FA0759, containing plasmid pFA0759, encoded a polypeptide of 187amino acids with 72% amino acid sequence identity to a putative subunitof the vacuolar associated protein from Neurospora crassa (Tremblaccession number P87252). This gene product did not appear to be asecreted protein as no signal peptide was detected using Signal-Psoftware. Clone FA0759 was represented by approximately 2.0% of the cDNAclones in Library A and 1.5% of Library B.

Example 10 Cloning and Nucleotide Sequence Analysis of Genomic DNASegments Encoding Unknown Genes “Daria” and the Putative VacuolarAssociated Protein

Fusarium venenatum genomic DNA segments encoding the unknown secretedprotein “Daria” and the putative vacuolar associated protein wereisolated by screening the λZipLox library described in Examples 6 and 7.Radiolabeled cDNA inserts from plasmids pFA0035 and pFA0759 were used asprobes to screen the library. Plaques which hybridized strongly toeither probe using the same conditions in Example 7 were purified twiceon E. coli Y1090ZL cells, and the individual clones were subsequentlyexcised from the λZipLox vector as pZL1-derivatives (D'Alessio et al.,1992, supra).

DNA sequence analysis revealed that one of these clones, containing aplasmid designated as pECO3, comprised the entire “Daria” coding regionas well as 0.9 kb of 5′-flanking and 0.9 kb of 3′-flanking DNAencompassing the promoter and terminator regions, respectively. Thenucleotide sequence (SEQ ID NO:2) and deduced amino acid sequence (SEQID NO:5) are shown in FIG. 2. The coding region was punctuated by asingle 55 bp intron.

Similarly, a genomic DNA clone was isolated through screening of theλZipLox library with a radiolabeled probe derived from the cDNA insertof pFA0579. The genomic clone, designated as pQUINN, encoded the entirevacuolar associated protein subunit plus 3.0 kb of 5′-flanking and 0.3kb of 3′-flanking DNA comprising the promoter and terminator regions,respectively. The nucleotide sequence (SEQ ID NO:3) and deduced aminoacid sequence (SEQ ID NO:6) are shown in FIG. 3. Nucleotide sequencecomparisons between the genomic and cDNA clones revealed the presence ofa 594 bp intron in the 5′-untranslated region of the putative vacuolarassociated protein subunit. In addition, the coding region contained a77 bp intron.

Example 11 Construction of pDMI81

Plasmid pDM181 was constructed using the technique of splice overlapextension to fuse the 1.2 kb Fusarium oxysporum trypsin promoter to the1.1 kb Fusarium oxysporum trypsin terminator. A polylinker containingSwaI, KpnI and PacI restriction sites was inserted between the promoterand terminator as part of the overlapping PCR strategy. At the 5′ end ofthe promoter a XhoI site was added and the native EcoRI site waspreserved. At the 3′ end of the terminator EcoRI, HindIII and NsiI siteswere incorporated by the PCR reaction.

A PCR fragment containing −1208 to −1 of the Fusarium oxysporum trypsinpromoter plus a 25 base pair polylinker was generated from plasmidpJRoy20(Royer et al., 1995, Biotechnology 13: 1479-1483) using thefollowing primers:

Primer 1 (sense): 5′-GAGCTCGAGGAATTCTTACAAACCTTCAAC-3′ (SEQ ID NO:7)       XhoI   EcoRI Primer 2 (antisense):5′-TTAATTAAGGTACCTGAATTTAAATGGTGAAGAGATAGATATCCAAG-3′ (SEQ ID NO:8)    PacI    KpnI      SwaI

The 100 μl PCR reaction contained 10 ng of pJRoy20, 50 pmol of eachprimer, 1×Pwo buffer (Boehringer Mannheim, Indianapolis, Ind.), 200 μMeach of dATP, dCTP, dGTP, and dTTP, and 5 units of Pwo DNA polymerase(Boehringer Mannheim, Indianapolis, Ind.). PCR conditions used were 95°C. for 3 minutes followed by 25 cycles at 95° C. for 30 seconds, 50° C.for 1 minute, and 72° C. for 1 minute. The final extension cycle was at72° C. for 5 minutes.

Using the same PCR conditions, a second PCR fragment containing basepairs −5 to −1 of the Fusariun oxysporum trypsin promoter, a 25 basepair polylinker, and 1060 base pairs of the 3′ untranslated region ofthe Fusarium oxysporum trypsin gene (terminator region) was generatedfrom plasmid pJRoy2O using the following primers:

Primer 3 (sense):5′-TCACCATTTAAATTCAGGTACCTTAATTAAATTCCTTGTTGGAAGCGTCGA-3′ (SEQ ID NO:9)          SwaI     KpnI    PacI Primer 4 (antisense):5′-TGGTATGCATAAGCTTGAATTCAGGTAAACAAGATATAATTT-3′ (SEQ ID NO: 10)       NsiI HindIIIEcoRI

The final 2.3 kb overlapping PCR fragment which contained −1208 to −1 ofthe Fusarium oxysporum trypsin promoter, the 25 base pair polylinker and1060 base pairs of the Fusarium oxysporum trypsin terminator wasobtained using 0.2 μl of the first PCR (promoter) reaction and 3 μl ofthe second (terminator) reaction as templates and primers 1 and 4. ThePCR conditions used were 95° C. for 3 minutes followed by 30 cycles at95° C. for 30 seconds, 62° C. for 1 minute, and 72° C. for 3 minutes.The final extension cycle was at 72° C. for 5 minutes. Pwo DNApolymerase was also used for this reaction.

The resulting 2.3 kb fragment containing the trypsin promoter, thepolylinker, and the trypsin terminator was digested with EcoRI andligated into the EcoRI digested vector pMT1612 containing the bar gene(WO 97/26330) to create pDM181 (FIG. 4).

Example 12 Construction of plasmid pSheB1

The Fusarium venenatum expression vector pSheB1 (FIG. 5) was generatedby modification of pDM181. The modifications included (a) removal of twoNcoI sites within the pDM181 sequence, and (b) restoration of thenatural translation start of the Fusarium oxysporum trypsin promoter(reconstruction of an NcoI site at the ATG start codon).

Removal of two NcoI sites within the pDM181 sequence was accomplishedusing the QuikChange™ site-directed mutagenesis kit (Stratagene CloningSystems, La Jolla, Calif.) according to the manufacturer's instructionwith the following pairs of mutagenesis primers:

5′-dCAGTGAATTGGCCTCGATGGCCGCGGCCGCGAATT-3′ plus (SEQ ID NO:11)

5′-dAATTCGCGGCCGCGGCCATCGAGGCCAATTCACTG-3′ (SEQ ID NO:12)

5′-dCACGAAGGAAAGACGATGGCTTTCACGGTGTCTG-3′ plus (SEQ ID NO:13)

5′-dCAGACACCGTGAAAGCCATCGTCTTTCCTTCGTG-3′ (SEQ ID NO:14)

Restoration of the natural translation start of the Fusarium oxysporumtrypsin promoter was also accomplished using the Stratagene QuikChange™site directed mutagenesis kit in conjunction with the following pair ofmutagenesis primers:

5′-dCTATCTCTTCACCATGGTACCTTAATTAAATACCTTGTTGGAAGCG-3′ plus (SEQ IDNO:15)

5′-dCGCTTCCAACAAGGTATTTAATTAAGGTACCATGGTGAAGAGATAG-3′ (SEQ ID NO:16)

All site-directed changes were confirmed by DNA sequence analysis of theappropriate vector regions.

Example 13 Construction of pDM194 and pDM218

The 7.8 kb plasmid pDM194 (7.8 kb) used to obtain expression ofThermomyces lanuginosus (formerly designated Humicola lanuginosa )lipase in Fusarium venenatum has the following gene elements:

A 1.2 kb DNA segment containing the Fusarium oxysporum trypsin genepromoter (Royer et al., 1995, supra).

A 0.9 kb DNA fragment containing the Thermomyces lanuginosus lipase cDNA(EP 305 216).

A 1.1 kb DNA segment containing the Fusarium oxysporum trypsin geneterminator (Royer et al., 1995, supra).

A 4.7 kb fragment of DNA from pMTI612 (WO 98/11203) containing the 2.8kb E. coli vector pUC19 and a 1.8 kB fragment comprising the Aspergillusnidulans amdS gene promoter (Hynes et al., 1988, Mol. Cell. Biol. 8:2589-2596), Streptomyces hygroscopicus phosphinothricinacetyltransferase (bar) gene (Thompson et al., 1987, EMBO Journal 6:2519-2514), and Aspergillus niger AMG terminator.

The SwaI/PacI lipase fragment was generated by PCR. Plasmid pMHan37which contains the cDNA of the Thermomyces lanuginosus lipase gene (EP305 216) was used as the template. PCR primers 5 and 6 shown below wereused to introduce a SwaI site at the 5′ end and a PacI site at the 3′end of the lipase coding region.

Primer 5 (sense): (SEQ ID NO: 17) 5′-ATTTAAATGATGAGGAGCTCCCTTGTGCTG-3′     SwaI Primer 6 (anti-sense): (SEQ ID NO: 18)5′-TTAATTAACTAGAGTCGACCCAGCCGCGC-3′      PacI

The 100 μl PCR reaction contained 10 ng of pMHan37, 50 pmol of eachprimer, 1×PCR buffer (Perkin-Elmer Corp., Branchburg, N.J.), 250 μM eachof dATP, dCTP, dGTP, and dTTP, and 5 units of Taq DNA polymerase(Perkin-Elmer Corp., Branchburg, N.J.). The PCR conditions used were onecycle at 95° C. for five minutes followed by 30 cycles at 95° C. for 1minute, 55° C. for one minute, and 72° C. for two minutes. The 0.9 kbPCR product was subcloned into pCRII of the TA Cloning Kit (Invitrogen,Carlsbad, Calif.) according to the manufacturer's instructions. PlasmidDNA was isolated using a Qiagen Maxiprep Kit (Qiagen, Santa Clarita,Calif.), digested with SwaI and PacI, and electrophoresed on a 1%agarose gel for one hour at 100 volts. The 0.9 kb fragment was excised,purified using a SpinBind Kit (FMC, Rockland, Me.), and cloned intopBANe6 (WO 98/11203) to produce pBANe8.

pBANe8 was digested with SwaI and PacI and the 0.9 kb lipase fragmentwas ligated to SwaI/PacI digested pDM 181 yielding lipase expressionplasmid pDM 194 (FIG. 6). pDM218 was constructed from pJRoy35 (FIG. 7).NotI and PmeI restriction sites were introduced at the 5′ end of theFusarium oxysporum trypsin promoter by PCR. A 362 bp amplicon containingthe 5′ end of the promoter was produced using pDM194 as a template withthe following primers:

multi1: 5′-ATAAGAATGCGGCCGCTAGTTTAAACTTACAAACCTTCAACAGTG-3′ (SEQ IDNO:19)

multi 2: 5′-TAGCATCTATCTCCGTCTT-3′ (SEQ ID NO:20)

The 100 μl PCR reaction contained 150 ng of 3 kb EcoRI fragment, 50 pmolof each primer, 1×Pwo buffer, 200 μM each of dATP, dCTP, dGTP, and dTTP,and 5 units of Pwo DNA polymerase. The PCR conditions used were onecycle at 95° C. for 3 minutes, 30 cycles at 95° C. for 1 minute, 50° C.for 1 minute, and 72° C. for 1.5 minutes; followed by a 5 minuteextension at 72° C. The amplicon was subcloned into vector pCR-Blunt(Invitrogen, Carlsbad, Calif.). A 0.25 kb NotI/BcuI fragment waselectrophoresed on a 1% agarose gel for one hour at 100 volts, excised,and purified using a QIAquick Gel Extraction Kit (Qiagen, Santa Clarita,Calif.).

HpaI, SnaBI, and PpuMI sites were introduced at the 3′ end of theFusarium oxysporum trypsin terminator by PCR. A 714 bp ampliconcontaining the 3′ end of the trypsin terminator was produced using the 3kb EcoRI fragment as template with the following primers:

multi3: 5′-GTGTGCAGTGACCCAGAAT-3′ (SEQ ID NO:21)

multi4: 5′-GATTGGGTCCCTACGTAGTTAACACTATAGGCCATCGTTTAC-3′ (SEQ ID NO:22)

The 100 μl PCR reaction contained 50 pmol of each primer, 150 ng of the3 kb EcoRI fragment, 1×Pwo buffer, 200 μM each of dATP, dCTP, dGTP, anddTTP, and 5 units of Pwo DNA polymerase. The PCR conditions used wereidentical to those listed above. The amplicon was subcloned into vectorpCR-Blunt. A 0.62 kb NheI/PpuMI fragment was isolated as describedabove.

A 2.3 kb BcuI/NheI fragment containing the 3′ end of the Fusariumoxysporum trypsin promoter, the Humicola lanuginosa lipase gene (Geneseqnucleotide accession number N91076), and the 5′ end of the Fusariumoxysporum trypsin terminator was isolated from pDMI94.

The 0.25 kb NotI/BcuI fragment, 2.3 kb BcuI/NheI fragment, and 0.62 kbNheI/PpuMI fragments were cloned together into pDMI94 partially digestedwith NotI and completely digested with PpuMI to create pDM218 (FIG. 8).

Example 14 Construction of Plasmid pMWR60

The plasmid pMWR60 was derived from the expression vector pEJG25A.pEJG25A was constructed by insertion of a phytase coding sequence fromPeniophora lycii (WO 98/28408) into pDM181 (Example 11) as follows:

First, two synthetic oligonucleotide primers shown below were designedto amplify the Peniophora lyci phytase coding sequence from plasmidpAlphy2 (WO 98/28408) as template by PCR.

Forward primer: 5′-ATTTAAATATGGTTTCTTCGGCATTCGC-3′ (SEQ ID NO:23)

Reverse primer: 5′-TTAATTAACTATTCCGACGGAACAAAGC-3′ (SEQ ID NO:24)

(Bold letters represent coding sequence).

Each 100 μl Pwo polymerase reaction contained 50 pmol of each primer, 1ng of template DNA, 2 μl of 10 mM dNTPs, 1×Pwo polymerase buffer, and2.5 units of Pwo polymerase. The reactions were incubated in a PerkinElmer Model 9600 Thermal Cycler programmed as follows: One cycle at 94°C. for 2 minutes, 55° C. for 30 seconds, and 72° C. for 1 minute; 9cycles at 94° C. for 15 seconds, 55° C. for 30 seconds, and 72° C. for 1minute; 15 cycles at 94° C. for 15 seconds, 55° C. for 30 seconds, and72° C. for 1 minute, with an extension of 20 seconds per cycle; a finalcycle at 94° C. for 15 seconds, 55° C. for 30 seconds, and 72° C. for 7minutes; and a soak cycle at 4° C. The reaction product waselectrophoresed on a 1% agarose gel for one hour at 100 volts. The 1.3kb band was excised and purified using Qiaex II. The purified PCRproduct was subsequently cloned into the plasmid pCR2. 1 (Invitrogen,Carlsbad, Calif.) and used to transform E. coli TOP10 cells (Invitrogen,Carlsbad, Calif.). Plasmid DNA was isolated from several of thetransformant colonies and analyzed by DNA sequencing to ensure that nomutations were introduced during the PCR. Subsequently one phytase clonewas digested with restriction endonucleases SwaI and PacI. The fragmentswere electrophoresed on a 1% agarose gel for one hour at 100 volts,excised, and purified using Qiaex II. The 1.3 kb phytase gene fragmentwas ligated with pDM181 (Example 11) that had been previously cut withSwaI and PacI, resulting in the expression plasmid pEJG25A (FIG. 9).

In order to remove extraneous linker sequences in the 5′-flanking DNA,pEJG25A was mutagenized using the Quick-Change™ site-directedmutagenesis kit in combination with the following oligonucleotideprimers to obtain the vector pMWR60-Int2:

primer A: 5′-CTCTTGGATATCTATCTCTTCACCATGGTTTCTTCGGCATTCGC-3′ (SEQ IDNO:25)

primer B: 5′-GCGAATGCCGAAGAAACCATGGTGAAGAGTAGATATCCAAGAG-3′ (SEQ IDNO:26)

The designed nucleotide changes were verified by DNA sequence analysis.

pMWR60-Int2 was then digested with SfuI plus NheI, and the 1.8 kbfragment was electrophoresed on a 1% agarose gel for one hour at 100volts, excised, and purified using Qiaex II. The isolated fragment wasligated with the SfuI-NheI vector fragment of pDM218 to generate a newintermediate called pMWR60-Int3. This intermediate was cleaved withNotI, and a 5.35 kb fragment was purified as above. The purifiedfragment was ligated with pSheB1 (described in Example 12) which hadbeen previously digested with NotI. The resulting intermediate wasdesignated as pMWR60-Int4a. pMWR60-Int4a was then cut with BspLU11I plusNheI, and a 5.25 kb segment was purified as above. This isolated segmentwas ligated with pSheB1, which had also been digested with BspLU11I plusNheI, and purified as above. The resulting vector product was namedpMWR60 (FIG. 10).

Example 15 Generation of a Lipase Reporter Gene

For the construction of variants of a Humicola lanuginosa lipase knownas LIPOLASE™ (Novo Nordisk A/S, Bagsvaerd, Denmark), the Chameleondouble-stranded, site-directed mutagenesis kit was used according to themanufacturer's instructions.

The gene encoding the LIPOLASE™ enzyme was obtained from pAHL (WO92/05249). In accordance with the manufacturer's instructions, the ScaIsite of the ampicillin gene of pAHL was changed to a MluI site by use ofthe primer 7258, thus changing the ScaI site found in the ampicillinresistance gene and used for cutting to a MluI site. Primer 7258:5′-GAATGACTTGGTTGACGCGTCACCAGTCAC-3′ (SEQ ID NO:27)

The pAHL vector comprising the LIPOLASE™ gene was then used as atemplate for DNA polymerase with oligos 7258 and 7770, thus changing theScaI site found in the LIPOLASE™ gene and without changing the aminoacid sequence site. Primer 7770: 5′-TCTAGCCCAGAATACTGGATCAAATC-3′ (SEQID NO:28)

The desired mutation (e.g., the introduction of a cysteine residue) wasintroduced into the LIPOLASE™ gene by addition of an appropriate oligoscomprising the desired mutation.

Site directed mutagenesis as described above was used to construct aplasmid harboring a gene encoding the LIPOLASE™ variant IS, E239C, Q249Rusing the following primers:

Primer 106659 shown below was used to introduce E99N,N101S.

Primer 107581:

5′-CTTAACTTTGACTTGAAAAACATATCTGACATTTGCTCC-3′ (SEQ ID NO:29)

Primer 101782 shown below was used to introduce SPPCGRRP (-E).

Primer 101782:

5′-GGACGGCCTTGGCTAGCCCTCCGTGCGGCCGCCGGCCGGTCTCGCAGGATCTGT TTAAC-3′ (SEQID NO:30)

Primer 9639 shown below was used to introduce E239C.

Primer 9639: 5′-ATATCGTGAAGATATGCGGCATTGATGCCACC-3′ (SEQ ID NO:31)

Primer 8829 shown below was used to introduce Q249R.

Primer 8829: 5′-GGCGGCAATAACCGGCCGAACATTCCGGATATCCC-3′ (SEQ ID NO:32)

The mutations were verified by sequencing the entire gene. The resultingplasmid was designated pEVi1163.

Example 16 Construction of pRaMB60

Plasmid pRaMB60 was constructed by ligating the 6.5 kb SfuI-NheI vectorfragment from pMWR60 with a 0.5 kb SfuI-NheI fragment from pSheB1. Allfragments were isolated by agarose gel electrophoresis and purified fromgel slices using BioRad Prep-a-Gene reagents (BioRad Laboratories,Hercules, Calif.).

Example 17 Construction of Plasmid pRaMB62, an Expression VectorContaining the Glucoamylase Promoter

A unique BspLU11I site was generated by site-directed mutagenesis ofplasmid pFAMG using the following primers in combination with theQuick-Change™ mutagenesis kit:

primer 1: 5′-dCACTGCTATCACCAACATGTTTACTCAAGTCC-3′ (SEQ ID NO:33)

primer 2: 5′-dGGACTTGAGTAAACATGTTGGTGATAGCAGTG-3′ (SEQ ID NO:34)

The site-directed change was verified by DNA sequencing, and theresulting plasmid derivative was designated pMWR62-Intl.

Next, the lipase reporter gene described in Example 15 was amplified byPCR using plasmid pEVi 1163 as template with the following primers whichintroduced a BspHI site near the start codon and a PacI site followingthe stop codon of the lipase coding region:

forward primer: 5′-GACTCATGAGGAGCTCCCTTGTGCTGTTC-3′ (SEQ ID NO:35)

reverse primer: 5′-TGATTAATTAACCTAAAGACATGTCCCAATTAAC-3′ (SEQ ID NO:36)

The PCR reaction was composed of 1 μl of template DNA (10 ng), 1 μl offorward primer (77 pmol), 1 μl of reverse primer (81 pmol), 10 μl of10×Pwo polymerase buffer, 16 μl of 1.25 mM dNTP mix, and 1 μl (2.5units) of Pwo polymerase. The reaction was incubated in a Perkin-ElmerModel 480 thermocycler using the following temperature settings: Onecycle at 95° C. for 5 minutes; 30 cycles at 95° C. for 1 minute, 47° C.for 1 minute, and 68° C. for 2 minutes; and a 4° C. soak cycle.

The amplified product was then digested with BspHI plus PacI, purifiedby agarose gel electrophoresis (Example 16), and used in a three-partligation with the 5.8 kb PmeI-NcoI fragment of pRaMB60 and the 2.1 kbStuI-BspLU11I fragment from pMWR62-intl. The resulting vector,designated pRaMB62 (FIG. 11), contained the lipase reporter gene underthe transcriptional control of the Fusarium venenatum glucoamylasepromoter.

Example 18 Construction of Plasmid pRaMB64, an Expression VectorContaining the “Daria” Promoter

The promoter region from pEC03 (Example 10), termed the “Daria”promoter, was amplified using PCR in combination with the followingprimer pair that were designed to introduce SwaI and BspLU11 I sites atthe 5′ and 3′ ends of the promoter segment, respectively:

primer 1: 5′-GCATTTAAATTACTACTGTGATGTG-3′ (SEQ ID NO:37)

primer 2: 5′-GATTGATGTGAAACACATGTTGATG-3′ (SEQ ID NO:38)

The PCR reaction was composed of 1 μl of pECO3 (10 ng), 1 μl of forwardprimer (50 pmol), 1 μl of reverse primer (50 pmol), 10 μl of 10×Pwopolymerase buffer, 16 μl of 1.25 mM dNTP mix, and 1 μl (2.5 units) ofPwo polymerase. The reaction was incubated in a Perkin-Elmer Model 480thermocycler using the following temperature settings: One cycle at 95°C. for 5 minutes; 30 cycles at 95° C. for 1 minute, 47° C. for 1 minute,and 68° C. for 2 minutes; and a 4° C. soak cycle.

The 0.9 kb PCR product was electrophoresed on a 1% agarose gel for onehour at 100 volts, excised, and purified using Qiaex II.

The amplified DNA segment was subcloned into pCR2.1 (Invitrogen,Carlsbad, Calif.) and analyzed by restriction enzyme cleavage with SwaI,BspLU11I, EcoRI, and XhoI to verify that it was correct. The plasmidgenerated in this manner, termed pECO4, was digested with SwaI plusBspLU11I, and the 0.9 kb promoter fragment was purified by gelelectrophoresis as described in Example 16. The purified fragment wasmixed in a three-part ligation with the 5.8 kb PmeI-NcoI fragment ofpRaMB60 and the 0.9 kb BspHI-PacI segment encoding the lipase reportergene. The product of this ligation, pRaMB64 (FIG. 12), contained the“Daria” promoter directing expression of the lipase reporter gene.

Example 19 Construction of Plasmid pRaMB66, an Expression VectorContaining the Promoter Derived from the Vacuolar Associated ProteinGene

The promoter segment from pQUINN (Example 10), encoding a putativevacuolar associated protein, was amplified by PCR with the followingprimers which introduced a SmaI site and an NcoI site at the 5′ and 3′ends of the promoter, respectively:

primer 1: 5′-dCGACCCGGGAATTAGAGAGGTTAGG-3′ (SEQ ID NO:39)

primer 2: 5′-dCGTATAACCCATGGTGGACTTGTCGGAC-3′ (SEQ ID NO:40)

The PCR reaction was composed of 1 μl of pQUNN (10 ng), 1 μl of forwardprimer (50 pmol), 1 μl of reverse primer (50 pmol), 10 μl of 10×Pwopolymerase buffer, 16 μl of 1.25 mM dNTP mix, and 1 μl (2.5 units) ofPwo polymerase. The reaction was incubated in a Perkin-Elmer Model 480thermocycler using the following temperature settings: One cycle at 95°C. for 5 minutes; 30 cycles at 95° C. for 1 minute, 47° C. for 1 minute,and 68° C. for 2 minutes; and a 4° C. soak cycle.

The 3.1 kb amplified DNA fragment was electrophoresed on a 1% agarosegel for one hour at 100 volts, excised, and purified using Qiaex II.

The 3.1 kb amplified DNA fragment was subcloned into pCR-Script(Stratagene, La Jolla, Calif.) to generate the intermediate plasmidpQUINN-promoterA. Two restriction fragments were isolated from thisplasmid; a 2.4 kb SmaI-NdeI fragment, and a 0.7 kb NdeI-NcoI fragment(together these segments span the entire promoter region). The isolatedfragments were combined in a four-part ligation with the 8 kb PmeI-NcoIfragment of pRaMB60 and the 0.9 kb BspHI-PacI segment encoding thelipase reporter gene described in the previous examples. The product ofthis ligation, pRaMB66 (FIG. 13), contained the lipase reporter geneunder the transcriptional control of the putative vacuolar associatedprotein gene promoter.

Example 20 Expression of the Lipase Reporter Gene in Fusarium venenatumUnder Control of the AMG, “Daria” and Vacuolar Associated ProteinPromoters

Spores of Fusarium venenatum CC1-3 (MLY-3) were generated by inoculatinga flask containing 500 ml of RA sporulation medium with 10 plugs from a1×Vogels medium plate (2.5% Noble agar) supplemented with 2.5% glucoseand 2.5 mM sodium nitrate and incubating at 28° C., 150 rpm for 2 to 3days. Spores were harvested through Miracloth (Calbiochem, San Diego,Calif.) and centrifuged 20 minutes at 7000 rpm in a Sorvall RC-5Bcentrifuge (E. 1. DuPont De Nemours and Co., Wilmington, Del.). Pelletedspores were washed twice with sterile distilled water, resuspended in asmall volume of water, and then counted using a hemocytometer.

Protoplasts were prepared by inoculating 100 ml of YEPG medium with4×10⁷ spores of Fusarium venenatum CC1-3 and incubating for 16 hours at24° C. and 150 rpm. The culture was centrifuged for 7 minutes at 3500rpm in a Sorvall RT 6000D (E. I. DuPont De Nemours and Co., Wilmington,Del.). Pellets were washed twice with 30 ml of 1 M MgSO₄ and resuspendedin 15 ml of 5 mg/ml of NOVOZYME 234™ (batch PPM 4356, Novo Nordisk A/S,Bagsvaerd, Denmark) in 1 M MgSO₄. Cultures were incubated at 24° C. and150 rpm until protoplasts formed. A volume of 35 ml of 2 M sorbitol wasadded to the protoplast digest and the mixture was centrifuged at 2500rpm for 10 minutes. The pellet was resuspended, washed twice with STC,and centrifuged at 2000 rpm for 10 minutes to pellet the protoplasts.Protoplasts were counted with a hemocytometer and resuspended in an8:2:0.1 solution of STC:SPTC:DMSO to a final concentration of 1.25×10⁷protoplasts/ml. The protoplasts were stored at −80° C., aftercontrolled-rate freezing in a Nalgene Cryo 1° C. Freezing Container (VWRScientific, Inc., San Francisco, Calif.).

Frozen protoplasts of Fusarium venenatum CC 1-3 were thawed on ice. Fiveto ten μg of pRaMB62, pRaMB64, and pRaMB66 (described in Examples 17-19)and 5 μl of heparin (5 mg per ml of STC) were added to separate 50 mlsterile polypropylene tubes. One hundred μl of protoplasts was added toeach tube, mixed gently, and incubated on ice for 30 minutes. One ml ofSPTC was added and incubated 20 minutes at room temperature. After theaddition of 25 ml of 40° C. COVE top agarose, the mixture in each tubewas poured onto an empty 150 mm diameter plate and incubated overnightat room temperature. Approximately 24 hours later, an additional 25 mlof 40° C. COVE top agarose containing 10 mg of BASTA™ per ml was pouredon top of the plate and incubated at room temperature for up to 14 days.The active ingredient in the herbicide BASTA™ is phosphinothricin.BASTA™ was obtained from AgrEvo (Hoechst Schering, Rodovre, Denmark) andwas extracted twice with phenol:chloroform:isoamyl alcohol (25:24:1),and once with chloroform:isoamyl alcohol (24:1) before use.

The transformants were picked directly from the selection plates (COVEunderlay with COVE-BASTA™ overlay) into 125 ml shake flasks containing25 ml of M400Da medium supplemented with 1 mM CaCl₂ and 100 μg/mlampicillin (to prevent bacterial contamination) and incubated at 28° C.,200 rpm on a platform shaker for 7 days. The untransformed recipientstrain was also included as a negative control.

Flasks were sampled at 7 days. Cells were removed by centrifugation, and10 μl of each supernatant sample was heated to 95° C. for 5 minutes withan equal volume of Tris-glycine sample buffer (Novex ExperimentalTechnology, San Diego, Calif.). The denatured supernatant proteins wereseparated on a 10-20% Tris-glycine gradient gel (Novex ExperimentalTechnology, San Diego, Calif.) and stained with Coomassie blue. SDS-PAGEanalysis showed that the lipase-producing transformants secrete aprominent polypeptide with an apparent molecular weight of approximately43 kDa.

Similarly, cell-free culture broths from each transformant were assayedfor lipase activity using p-nitrophenylbutyrate as the substrate (Royeret al., 1995, supra).

The results shown in Table I demonstrated that active lipase wasexpressed and secreted using the Fusarium venenatum promoter elementspresent in pRaMB62, pRaMB64, and pRaMB66.

TABLE 1 Lipase yield Vector used Promoter Transformant (LU/ml) None —negative control <1.0 pRaMB62 glucoamylase RaMB62.1  1158 ″ ″ RaMB62.2 500 ″ ″ RaMB62.3  1379 ″ ″ RaMB62.4  1678 ″ ″ RaMB62.5  702 ″ ″RaMB62.6  616 ″ ″ RaMB62.7  473 ″ ″ RaMB62.8  894 ″ ″ RaMB62.9  564 ″ ″RaMB62.10 1036 ″ ″ RaMB62.11 2731 ″ ″ RaMB62.12 1960 ″ ″ RaMB62.13 1682″ ″ RaMB62.14 572 ″ ″ RaMB62.15 1421 pRaMB64 “Daria” RaMB64.1  1217 ″ ″RaMB64.2  561 ″ ″ RaMB64.3  875 ″ ″ RaMB64.4  839 ″ ″ RaMB64.5  1449 ″ ″RaMB64.6  354 ″ ″ RaMB64.7  377 ″ ″ RaMB64.8  184 ″ ″ RaMB64.9  1967 ″ ″RaMB64.10 657 ″ ″ RaMB64.11 883 ″ ″ RaMB64.12 184 ″ ″ RaMB64.13 1935 ″ ″RaMB64.14 1049 ″ ″ RaMB64.15 875 pRaMB66 Vacuolar RaMB66.1  1990associated protein ″ ″ RaMB66.2  165 ″ ″ RaMB66.3  380 ″ ″ RaMB66.4  155″ ″ RaMB66.5  170 ″ ″ RaMB66.6  145 ″ ″ RaMB66.7  180 ″ ″ RaMB66.8  420″ ″ RaMB66.9  200 ″ ″ RaMB66.10 195 ″ ″ RaMB66.11 190 ″ ″ RaMB66.12 165″ ″ RaMB66.13 140 ″ ″ RaMB66.14 435 ″ ″ RaMB66.15 125

Example 21 Comparative Expression of the Humicola lanuginosa LipaseReporter Gene Under Control of Fusarium venenatum Amyloglucosidase andFusarium oxysporum Trypsin Promoters

Fusarium venenatum CC1-3 transformants containing either pRamB64(Example 17) or pDM218 (Example 13), prepared as described in Example20, were cultivated for 180 hours at 29° C., 1200 rpm, pH 6.25 in 2liter fermentors containing suitable carbon, nitrogen, and trace metalssources and supplemented with either urea or ammonium phosphate as thefeed. Cell-free culture broths from each fermentation were assayed at 18to 24 hour intervals for lipase activity using p-nitrophenylbutyrate asthe substrate (Royer et al., 1995, supra) for 180 hours.

FIG. 14 shows comparative expression of the Humicola lanuginosa lipasereporter gene in Fusarium venenatum under control of either the Fusariumvenenatum amyloglucosidase (pAMG) promoter or Fusarium oxysporum trypsin(pSP387) promoter. The results demonstrated higher levels of lipaseactivity with the Fusarium venenatum amyloglucosidase promoter than theFusarium oxysporum trypsin promoter whether urea or ammonium phosphatewas used as the source of nitrogen in the feed. Moreover, higher levelsof lipase activity were observed using ammonium phosphate in the feed

Deposit of Biological Materials

The following biological materials have been deposited under the termsof the Budapest Treaty with the Agricultural Research Service PatentCulture Collection, Northern Regional Research Center, 1815 UniversityStreet, Peoria, Ill., 61604, and given the following accession numbers:

Deposit Accession Number Date of Deposit E. coli TOP10 (pECO3) NRRLB-30067 Oct. 27, 1998 E. coli TOP10 (pFAMG) NRRL B-30071 Oct. 27, 1998E. coli DH10B (pQUINN) NRRL B-30076 Oct. 27, 1998 E. coli TOP10(pFB0346) NRRL B-30073 Oct. 27, 1998

The strains have been deposited under conditions that assure that accessto the culture swill be available during the pendency of this patentapplication to one determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C.§122. The deposits represent substantially pure cultures of thedeposited strains. The deposits are available as required by foreignpatent laws in countries wherein counterparts of the subjectapplication, or its progeny are filed. However, it should be understoodthat the availability of a deposit does not constitute a license topractice the subject invention in derogation of patent rights granted bygovernmental action.

The invention described and claimed herein is not to be limited in scopeby the specific embodiments herein disclosed, since these embodimentsare intended as illustrations of several aspects of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

40 1 6050 DNA Fusarium 1 aatttcgtcg atagcgaggg actcctggcc ctcgaatttagttagcgtat cagtgtaaag 60 tgctgggttc tccaggcgta agtaaattga accagatgttagctcccaga tttcgccccg 120 aagccggttg ggcagaccaa cgcggataag tttatggaaagttggttggc ggatcaatgt 180 aaagttcctg ccattgtcac gcagatactc ggcccaaagacgcatctttg ccctatcgcg 240 caactttttt gggtccccag gatatcggaa gagcattcctaagccagcat ctggtgggag 300 atcgttcttc ttatcttcgg ttcttaaaag atgttcagagtaacactcag cagcaactcg 360 acgcaacttg gctacgttgc caacgcctgc tctaagacccttcttgaggc cgtcacagaa 420 acgttcgcag gactgtctgg ttccagcgag atggattgttatgcgttgtt ctcgggggtc 480 tctcttgtct ttagaggtat cggcaagaag gccattccacgtagtcagag cgagtgcgaa 540 ctataaccag gcaacgtcag aatttgtacc atgcaagatttttataccac atacctggaa 600 gttctggcta ttcaagcgct ctacccttcg tatagcacataaaggaaatg tgaatccatt 660 gccactgggt cctcctccgt gtgtctggcc ggtaaaagcggtcgaggttg aaaggctagc 720 agattgcaca aagctagtgg gtgttgttga gaagcacatgtaatgctctg acaggtgtag 780 cttgccagca taatgccatc ctcgatcgtg atccttatcaccgtgagtag cgttggaggg 840 cgggatagta agctcggcgt tgatctcgta gaggggcgcctgagaggcgg gcaagcgaaa 900 ttggctctga aatagcgatg actttgaggg attccgatctgaactggata gattgagccc 960 ctgggtagga tcgataagct gctgggcttt ttgaacgatgttggtgaagt tcgaaaacat 1020 gatttcggtc agcggcgcat gacgaggggg gttccggtccaggagggagg tcgcggctga 1080 gcttgaagga gatgcaagac acgaagcgaa agacacgaagagagcgcaag agtctgagta 1140 tgtgcaacca ggctcgaata agtgcaaggc aggcagaagtacggaataga cgatagaatt 1200 gagtatagaa aggctgaatg gaagatggag acgagttataggacggtgga gatagagtgg 1260 agttgaagtt gaacgaagct gcgtcaggtc cagatacgggagactggcca tcaactactg 1320 gccaggtagc cagggcgcga tgggcgggtg ggcagggtcgcgggggggac ctcagggcat 1380 tcctttctcc aagggccgct ggggctatgg acggggctggctgaactcca gccgtcatgg 1440 gatagcggtg caagagatca ggtactaagt ctaccatgataatttagggg gcagagaaaa 1500 atgatatatt tgtttagtag taagcgggtt tttacagttgaggaaccaac cttcttcatt 1560 tatttattct ttctttctct gcaattcagt cctttttcttaaatagaata tctaccaatg 1620 gaacggcgtg gctgaagtgg ctgaagaata tagctcgagctgtcaaaccg ctcatcctac 1680 taccctaggt ataaagctgg gaactaagac tcatttctatccaactcatc atattgggag 1740 ttagtgtaga cctgtcggcc tagagaatat gtgtatctgcatactttcaa ataccctacg 1800 tatacccact atgtttagca caatcattga cctctcaaggcctcacccat ctcaacacct 1860 gtcgtgtgct cacttgacta cttctttgaa ccagctcgccatcggactag tcgaacaagc 1920 ttgtcgcccc catacagatg aatgtatgtt taaagctacatgatcagcct gaaccgagca 1980 taactcgagt gccgagactc ctctgatgta tatcgagatgaatgacaaac ctacgggtcc 2040 gttcttgaga agtggcctga gatttctcac ttggtgagaaaaaggacggg cgagcgggag 2100 cctgagtcag aagaaatacc tgtctccttg gatctcacatgacggtgttg tggaagagtg 2160 catctattgt cattgctgga gtgacggcag agtaggggtctaaagaaacc catactgagt 2220 agagatggag aagacaacaa aagcccaaga cgacagagacgacagaagat taaagctatc 2280 agagcgagac tatatcacta ttcgaaacct gcgagtaatttaacaagaag tacacatcat 2340 cattgttatc aattcgacga agacatggtc gaaaattcttgcggtgtata tgtctgttgt 2400 atatgggcct gggcattgtt atttttcgcc gtctttatgtgtactaacac ttccattgat 2460 accccagaac aaaagatgaa cgcttaaaca gcaccaaaatcaggagaaga atggcgctgc 2520 tctaggtatg cttctgggat aaaaagcgat gttgatacctctcagaaaag aagtgatttg 2580 aagttgaatc aaacaaatag ccgatggagc gatctgaaggggtggcagac ctgctacgcg 2640 catttaggca aggcatcaac tcggcagatg attaagaaaggttttgtagg ttcacgtgtt 2700 gtgttgtgtt ccattataag tttataacct tgctaagatgcaacgactct gacctcaggg 2760 tgttagaaaa attgaccact aggagcataa gtgacgaaattcggggatca agacaataga 2820 tagtttcatt ttcatgtgct cctacgtctt ttcacgtaatgtttcttata aaaaaaaaga 2880 tagcattgtc tctttggtga aaagagaaaa aaagatgttacgacgtggcc ttgattcgaa 2940 cagacgcctc cgaagagaat agatttctag tctatcgcgttagaccactc cgccaccacg 3000 ccttacgtaa tctgtgattg ttgaaagtta ctctcgtgttacggtctata cgtgaagaat 3060 ctacacttga cgagtctcga ggtctggggt cagttagacggaaatgggag aacaaagaga 3120 cttggtgaca ttgcaggcaa ccgggtagat gttgaggtcattgatcggac aagattgttg 3180 cttcaaaagt aacaggtatt ctttttttta atcaacagaaacgttccatg ttcatttgtt 3240 aatccaatct atttgtgata gcgtttgatg acaaacaataataatgatgg tctggcggct 3300 agtgatcgtt tgtaatgacg tcgtcatata tcctatcactatacagttgc tttgcacacg 3360 cactcacgtc cttcattcgt tgtcttcact atttgatggtgatttggttc aacaacctac 3420 agaaataatg acctgtggtg ttctccgaat atggctagaccaacacaagc ttgtaccgcg 3480 gcattcaaat caccatgtga tgcccatcat cagatcatccaccaacccaa aaacagacca 3540 actactcaca aaaaggcatc tcatcaagaa aaaacggccaactaacgtcc aaaaggcccg 3600 aaaaacgtcc atcacgccgc agccgagact tcaatagactgcacaagaag gaccgatgag 3660 atcgaccaga ctaaacccgg gagagtgtca aatatgcgggggattgggga acttacccca 3720 gaaaagagaa ggaggataaa ttccatgtct ggggttgacgtctctattgg ttagacacga 3780 acgcctgctc tcggcgtaat ttataccata gcgccaatgagggcggaaac tcctgttttg 3840 tcaagtcgtc attgttggtt gggtcatgat atatagccagtaggtatccg tcttggtgat 3900 tgaccagaca tatcgctcat cacagatcaa catcactgctatcaccaaca tgcttactca 3960 agtcctttat ggcttggtag ccagtgccct ttggcaaggccaagtcgttg catcaccaag 4020 caaggacaat tcactggagc gcttcattga caaacaagctgatatttcta tcaagggtgt 4080 ccttgctaat attggcgctg atggaaaaag ggcacagggtgcagcgcctg gtgctgttgt 4140 ggcaagtcca tcgaaagaag atcctgattg taagccagcatcctaccttg tccttgtccg 4200 catgctaatg atggtctcag attggtacac ttggactcgtgactctgctt taacgtacaa 4260 agtgctcgtt gagagattca tccacggcga caaatctctccaacgaaaga tagatgaata 4320 tgtctccgca caagcgaaac tgcaagggac cacaaatccatcgggcagcc cagagtcggg 4380 cggtctcggc gagccaaagt tccatgtgaa tctcactgctttcactggat cttggggtcg 4440 gcctcagcgc gacggccctc cgcttcgggc taccgccttgactctgtatg cagaatggct 4500 catttcccac ggcgaaagat ccaaggcttt gaacaaagtctggccagtca tcgagaagga 4560 ccttgcgtat actaccaagt tctggaatcg cactggctatgatctatggg aggaggttaa 4620 tggatcttct ttctttacac tttcggcttc gcatcgtgctcttgtcgaag gtgccgctct 4680 ggctaagaaa cttggcaaat cttgtcctga ctgtgtcaccaacgctcctc gcgttctgtg 4740 cttccttcag actttctgga ctggtggcta cgttgactccaacattaacg tcaaggatgg 4800 tcgcaagggt ctcgatgtca actccatcct ctcgtccattcatacattcg atcccaactc 4860 caagtgcacc gactcgacgt tccagccttg ttcacccagagctcttgcga accacaaggc 4920 ggtcgtcgat tctttcaggt caatctatgg tgtcaacaagaatagaggtc aaggcaaggc 4980 cgcggctgtt ggtcgatata gcgaggacgt gtactatgatggcaaccctt ggtacctggc 5040 cactcttgct gctgcagaac aactctacgc tgcggtctaccagtgggata agcttggcgc 5100 tgttactgtt gacgatgtat ctttgtcttt cttcaaggatatcgttccca aggtctccaa 5160 aggcacttat gccaagaaga ccaagacata caaggagatcatcaaagcag ccaagactta 5220 cgccgacggc tttgtcgctg tcgtgcagac atacactcccaaggacggct cactagctga 5280 gcaatttgac aagtcaactg gagcccccaa gtccgctgttcacctcacct ggtcctacgc 5340 cgcctttgtc gccacaactg aacgtcgcga cggcatcatctctccctcct ggggcgaaag 5400 cagcgccaac aaggtccccg ccgtgtgtca agctgccccagcatgtgaca caaccatcac 5460 cttcagtgtc aagaacgtgc aagtttcatc cgaccaaaaggtttacgtgg ttggctcagt 5520 gactgagctt tctaactggt cacctgatga tggcattgcgcttacgccat ctagttccgg 5580 agtgtggagc gtcaaggtta agattccttc tgatacaagctttgagtaca agtatatcaa 5640 gaagactagc agtggggatg ttacgtggtt gagtgatcccaacaaccggg ctattacggg 5700 tagcaagtgt ggaagtacaa gtactcttga tgatgagtggaggtagtgga tgacagattt 5760 atcaagctat gtagttttgt gaatatataa ttatccaaattatcagggtt cggtaagaat 5820 ataattcagt tcagcagtct gtacaagcaa gccatgattcacgcttcctt cgtttggaag 5880 ataagggttc ctcgccaccg tcaagataat tttctcgtgctaatatcacg taatccatct 5940 gcattaaacc cttgccggga aggtttcttc aacccagcaaccccagagta actcggagat 6000 agggaagtct atttgcctta tcctccgtga cgaattccctgaacccaatt 6050 2 2517 DNA Fusarium 2 aattactact gtgatgtgat cacacctaactaaataccta actcacccga tggatcgaca 60 aggaaatctc acgcccttgt cgagtctcctctttcgtctg tctcctgggc tcgctactgt 120 ccgattgtaa ctctcgctct ccaacttgttcaactctaat aagtggtggc acaacgtgaa 180 gatgtattgt tgtgtgaggc ggggggttgcgtggcattac caaagagacc aaaagtcccc 240 ctatgtcgat ttgatggtgt tgcgttgccatgatacggga ccccgaatat gttgtatgca 300 tcatgcgtac agaaagctac tgttcaaaacgaacggcaaa gcggattgat caacccgtga 360 aagaccatgg gtctctctca gtccacaatcttctcttcct gatcaaattt atggatccaa 420 gcggccacaa ttctagcgcc atcatgggtccctttcctct tttcgctcac cccatgttcc 480 ctgtcccacc tcattcagtg gacctgatggatccctatcc cccgatgagc cggggggtgc 540 agccttggcg ctctcttctt gttagtgtgacctactgttg atttcactca gcagtcctag 600 agtccattta gttgggcctg gggtgatggggtctgagact ttgcttcttg cctggtcttg 660 tctagctcga atctgtgggt tgcctggcctggcctggcct gacctgacct gaggggggtg 720 cccctttgct ctgttctgca tatgttgctattagctacct actcgagaat tcataaaagg 780 actgtccagc cccgtctctt actgacttctttcctttccc tcttcaccct cgttgtcata 840 tcaaatctgt cactcgttag accagactaccattcccact ttcgctttta aactacttta 900 ctcaactaat tctaatacca actccaaaaaccatcaacat gcgtttcaca tcaatcctcg 960 ctgccggcgc tttcgccacc atggccgctgcccagagcaa gaccgtctcc ctcgaccctg 1020 ctcagcagtc tcaggccgac tgcctctccgactgtgagcc tggcgatgtc aagtgccagt 1080 cttactgcat cactgtatgt tacaacaacgattcccctgt catgtgtaga aaactaacaa 1140 tcccaatagg ttccctctcc tgacgagaagaacatcgagg aaaccaccaa gtgttgtttg 1200 ccgcctgccc caagggcaag ggctccgaaggccgacactg agaagtacac cgtttgcatg 1260 aacgagtgta tcgccgacaa ctactggaagtccgttgatg gtaccccccg tggcaccgac 1320 gtccccgatg tcaagagcaa ggcctccgaggctgcctcct ccgctgctga gaaggccacc 1380 gccaccggta ctgctgctga gtctgatgctaccgccactg gtgcctccgc tactgagtcc 1440 gagtccggct ccgactccag ctccgaggagaccggctctg cctctggcac tgccactggt 1500 accgctgctg aggtctccga gactggtaacgccgcctctt ccctcgttgg tggtgtctcc 1560 ttcctcggtc tcgttgccgc tatcttcgctctgtaaattg ggtttcctgc tttaggataa 1620 tctgatttgg catgacggag aaggatttaatgggttttat tacagcggta atgattggag 1680 tttggatttc aagatgtgac acgttggacagcatgataag gcctacgggt ctgatcaatt 1740 tcatggacaa attttgtttt tttgggtaatcatttcgcgt tcacatatgg ctcggcatat 1800 gagcatgaat acaatacctc ttttttgcgcctcaattcat tccaatttct tgtgatctca 1860 cagtgattca acttacaagt tgcggcgcgaccactgaggt cgtgtctgat gtgggtcttc 1920 tgtttgtgat tggctcatga ttcccaatcgggtgcttcaa acgttagttt gtaaacaagc 1980 gaaatgaggg tcttaggatg catgttcaaagcgcaaaacc caattgaatt caaatgttaa 2040 agaatcatcg agaagagcga gttactgaggtgaatttgtg ctttcaactg tcaatacctc 2100 cctcagaaca aatgaattga attattattcacactcaatg cccaatattc taaacatgtt 2160 cgttgtaaca gagttttaat tccttgacgccacaatgttc cttggtaatt atcgcgcctg 2220 tcacatgaac tggctcctga acttaaacgttggtgaccca gcaactcgtt tatcaggctt 2280 agggtagctg tcatacaaca aacaaacttgtacaattgat gttattgatg aatcatgtat 2340 agaagagcac aattgattta aacacagataaactggtcga accgatttta tcaggttgtg 2400 tgaacatgca ttgccgaatc agaaaccagagtaagactat ctacagttcc atgaagacaa 2460 ttcacagact gccagaaagc aaggtactggaagcacgaga gacaaaatta ttgaatt 2517 3 4047 DNA Fusarium 3 aattagagaggttagggatt tcacatggcc accaatggga aggaggcaac catctgcacg 60 agcccaccaagtcatctcct caaactgtgc tgcgactaag aatttgattc cggttctggc 120 ctggcctttgtatcagctag gtcattctcg actaccggag gccaggctga agcagtcagt 180 cacgcattgtcactttatcg gtcctgtcct catacggata cactaggcgt caatgggctt 240 caaacggagatccagagatc tcatgaagag catcgacgat aaagtgagtg gttggggata 300 ctgtgcggtgccgaccccag cggcagccag gttccaccct tgattacatg gttgaaaagt 360 ggcgttactgggcgagatca aatttggcat gtatgttcgt ccaatgacgc gagctctcca 420 tgttgctgcgagggtcagga acggaccagc atgggatcag tgaggtgaaa tccaaccgag 480 ggagagcgagatctttgtgc tcatatccat gctgccatgc tacgtgccga acaggccaga 540 tggcgttcaactcagtcgac caggtccgat gaacgcggag cggtgacgag atcgaagctt 600 catctatcgcttacggggtt atgttccact ttccattaac gtttgcgagt tgctgtttga 660 gagccatgtcgaaagcatgg accgtgtcac atctttcaag gtaaatctgg aggtgggaag 720 aagaattgcgaggaacagga tgggaggata gcaggctgac gcggaaagct aggtagctac 780 ctggctgattactggctaaa gctggagagc aactaggtaa tatcaggcaa agagctccaa 840 gagctattgggaggctggct gattgtctct ggctgagacg caggaggaag ggttaaaatg 900 gccggcaggccaagaagggg ctgcaaaaca cggagtggat ggtggggcct cccacatacg 960 ggattcgggctgcggatcta acctcaattt ggcaagaggt aaataggacg acatgcaggc 1020 cccctgacatgtaaacaaga caagtggtaa acaagccatc aacatcaaca agagcgaaca 1080 atcgacacacccatgggggt gagatatggt agtaaggcag agagagatca gggcagcata 1140 cgtgggaaagggctgggcaa gaaaggacac aacggatcaa cagaacgcag cgctaccgag 1200 ggagcaacacaagtacagta accgctcaca gaggcacaac tcgtccaatc ctgcccccgt 1260 cttcaaaagcccagtttcgt tctgagtcct gtccggtccc tcttctcctc ctactccctc 1320 caattatcgccatccacatc gacatcgtca attcacaacc tcacccagac aagaagaaaa 1380 gaacgactgaaggccttcgc tcgccatcac ccgattcttt tccattctct tcgacttttg 1440 tttcgtaggaacaagagcca gagaacttct tgtcatcctt tcgaatttcg gaaggttgta 1500 tgagaagcttctctcgcgcc agcaaaagtc gcaaatctgg actttgaggc acgcgtcccc 1560 gttcccttcagcatcttccc atcgacatat cgggaatccg aatcccacac acagaccgtt 1620 accgaaacaaagatacacga agaggttgag atcaaacccc aacagcccga agccggacgg 1680 gaaggtgaaatatcttctgt ctccgtcacc gccgaacagg tccctcctcc tcgtcaagag 1740 caagagtttatcgaagaaga ggtccatatt acgcgtgaag aagaacatta ccaccgtccc 1800 ggtgtccaaaaattcgagca cgaagacttt actatccgtg aagactcccg acggtacgtt 1860 cgattttacatttcctttca tctccattta ggtcgcatct ttttcgttac ttttttggtc 1920 aattacacgggggatacgat tttcccacgg tcggagaaag ccctgcttgc tctctatgcc 1980 taggtctgtattctctcatc cctctgcgct gatctggcca tggagacgtg tgagaacaag 2040 actacaattcatcacatcat ttttcgctag gcgaaagcaa ttaccgttgt ccccgacctt 2100 ctcccaaccatcagttttca ctttcccttt tcttggtctg gcttgccttg accattaccc 2160 accgcgcacggagcgcttca gtccccagcc atcccattct cacatcactt ctcatatcct 2220 ctcttcacacgcctcacaca cccaccccct gcatgctacc atgccaaccc acttcagctt 2280 ggctggatacccaatttgct ttgcttcctc cccggctcac tagcgcctct aagcctgctg 2340 gcctgagcaaggcggtggag ctatctcagg ggccgccgcc tcccgttgcc atatgatacg 2400 caaacgacttactatagaca tccatcagct aacccagaca aatctagacc tcaacctccc 2460 tctcaataccaaccttccca gtaccaccaa ccttcccact accaaccacc tcccaaattc 2520 caaacttctcacactcacgt agagatcgac acccaccgtc atccctacta ctccaccccc 2580 attgatctcgctgaacgtga ataccgccag cgttaccgcc ctgcccaagc tttttccaca 2640 gaagacccttcttcccactc tcatcctcac taccaacctc aagacaactt caaagccaac 2700 aactacaccgttgaaggccg acccgctccc caattccatt cctctgagaa gactgaaatc 2760 aacaagtttactgttgacga acactcctct cgccctcagt acaaccacac cgagaagacc 2820 gaattcaacaactacactgt tgacagccga tcttcccgtc ctcaatacaa cacctgtgag 2880 aagactgagatcaacaattt cactgttgac gcccgctctt cccagccacg gtaccgcgac 2940 accaagacaactcaagtcaa cagctacgcc gttgacaagc ccgtttctcg tccatcttac 3000 aagaaggacgtgagatttac tgaacaaacc gtcgaagctt caaagtccga caagtccaag 3060 atgggttactacgacgacga gggtaagtga aatctgtcac ccagcgagcg ccatcaagct 3120 ctctattcgtgacgcaattc aagctaaccc agtcaccagg ttctttccgc aacggcggca 3180 tccacaagctcggtgacaag tcccgcgaca ttgaggttga cattcgcgag acttctcgtc 3240 ctgccaatgactgcgctccc aacaccgtca gcatcccctg ccaccacatc cgtctgggtg 3300 atttcctcatgctccagggc cgcccctgcc aggtcatccg catctccacc tcctctgcca 3360 ctggccagtaccgctacctt ggtgtcgacc tcttcaccaa gcagcttcat gaggagtctt 3420 ctttcatctccaaccctgcc cccagcgttg tcgttcagtc catgctcggc cctgtcttca 3480 agcagtaccgtgtcctcgat atgcaggagg gtcagatcgt tgccatgacc gagactggcg 3540 acgtcaagcagggtctccct gtcattgacc agtccaacct ctactctcgc ctccacaacg 3600 ctttcgagtccggtcgtggc tctgttcgcg tcctcgtcct caacgacggt ggccgtgagc 3660 ttgccgttgacatgaaggtc atccacggct ctcgcctgta agcgtgttca actgttttct 3720 gaattcgggcagccgcttgc aatgcgactt cttcccaatg tttaattgag tgaagggaca 3780 gcactaccagtctcacctca actgtgggga gcgggtctgg gctgtctcta atcttacctg 3840 tacaatgtcaagtttcatag gggacctgtt gtgtcaagat ggttcgagtt ttgtttgtgt 3900 caagattggataaatgatat tggctagctg gaaatactgg agtcttttgt gtagatggga 3960 gagttctgtacatgaactat agtaattgac aattgattcc gcatctactt agcttttcat 4020 tggtgctctatgcccaacat gtgaatt 4047 4 581 PRT Fusarium 4 Met Leu Thr Gln Val Leu TyrGly Leu Val Ala Ser Ala Leu Trp Gln 1 5 10 15 Gly Gln Val Val Ala SerPro Ser Lys Asp Asn Ser Leu Glu Arg Phe 20 25 30 Ile Asp Lys Gln Ala AspIle Ser Ile Lys Gly Val Leu Ala Asn Ile 35 40 45 Gly Ala Asp Gly Lys ArgAla Gln Gly Ala Ala Pro Gly Ala Val Val 50 55 60 Ala Ser Pro Ser Lys GluAsp Pro Asp Tyr Trp Tyr Thr Trp Thr Arg 65 70 75 80 Asp Ser Ala Leu ThrTyr Lys Val Leu Val Glu Arg Phe Ile His Gly 85 90 95 Asp Lys Ser Leu GlnArg Lys Ile Asp Glu Tyr Val Ser Ala Gln Ala 100 105 110 Lys Leu Gln GlyThr Thr Asn Pro Ser Gly Ser Pro Glu Ser Gly Gly 115 120 125 Leu Gly GluPro Lys Phe His Val Asn Leu Thr Ala Phe Thr Gly Ser 130 135 140 Trp GlyArg Pro Gln Arg Asp Gly Pro Pro Leu Arg Ala Thr Ala Leu 145 150 155 160Thr Leu Tyr Ala Glu Trp Leu Ile Ser His Gly Glu Arg Ser Lys Ala 165 170175 Leu Asn Lys Val Trp Pro Val Ile Glu Lys Asp Leu Ala Tyr Thr Thr 180185 190 Lys Phe Trp Asn Arg Thr Gly Tyr Asp Leu Trp Glu Glu Val Asn Gly195 200 205 Ser Ser Phe Phe Thr Leu Ser Ala Ser His Arg Ala Leu Val GluGly 210 215 220 Ala Ala Leu Ala Lys Lys Leu Gly Lys Ser Cys Pro Asp CysVal Thr 225 230 235 240 Asn Ala Pro Arg Val Leu Cys Phe Leu Gln Thr PheTrp Thr Gly Gly 245 250 255 Tyr Val Asp Ser Asn Ile Asn Val Lys Asp GlyArg Lys Gly Leu Asp 260 265 270 Val Asn Ser Ile Leu Ser Ser Ile His ThrPhe Asp Pro Asn Ser Lys 275 280 285 Cys Thr Asp Ser Thr Phe Gln Pro CysSer Pro Arg Ala Leu Ala Asn 290 295 300 His Lys Ala Val Val Asp Ser PheArg Ser Ile Tyr Gly Val Asn Lys 305 310 315 320 Asn Arg Gly Gln Gly LysAla Ala Ala Val Gly Arg Tyr Ser Glu Asp 325 330 335 Val Tyr Tyr Asp GlyAsn Pro Trp Tyr Leu Ala Thr Leu Ala Ala Ala 340 345 350 Glu Gln Leu TyrAla Ala Val Tyr Gln Trp Asp Lys Leu Gly Ala Val 355 360 365 Thr Val AspAsp Val Ser Leu Ser Phe Phe Lys Asp Ile Val Pro Lys 370 375 380 Val SerLys Gly Thr Tyr Ala Lys Lys Thr Lys Thr Tyr Lys Glu Ile 385 390 395 400Ile Lys Ala Ala Lys Thr Tyr Ala Asp Gly Phe Val Ala Val Val Gln 405 410415 Thr Tyr Thr Pro Lys Asp Gly Ser Leu Ala Glu Gln Phe Asp Lys Ser 420425 430 Thr Gly Ala Pro Lys Ser Ala Val His Leu Thr Trp Ser Tyr Ala Ala435 440 445 Phe Val Ala Thr Thr Glu Arg Arg Asp Gly Ile Ile Ser Pro SerTrp 450 455 460 Gly Glu Ser Ser Ala Asn Lys Val Pro Ala Val Cys Gln AlaAla Pro 465 470 475 480 Ala Cys Asp Thr Thr Ile Thr Phe Ser Val Lys AsnVal Gln Val Ser 485 490 495 Ser Asp Gln Lys Val Tyr Val Val Gly Ser ValThr Glu Leu Ser Asn 500 505 510 Trp Ser Pro Asp Asp Gly Ile Ala Leu ThrPro Ser Ser Ser Gly Val 515 520 525 Trp Ser Val Lys Val Lys Ile Pro SerAsp Thr Ser Phe Glu Tyr Lys 530 535 540 Tyr Ile Lys Lys Thr Ser Ser GlyAsp Val Thr Trp Leu Ser Asp Pro 545 550 555 560 Asn Asn Arg Ala Ile ThrGly Ser Lys Cys Gly Ser Thr Ser Thr Leu 565 570 575 Asp Asp Glu Trp Arg580 5 200 PRT Fusarium 5 Met Arg Phe Thr Ser Ile Leu Ala Ala Gly Ala PheAla Thr Met Ala 1 5 10 15 Ala Ala Gln Ser Lys Thr Val Ser Leu Asp ProAla Gln Gln Ser Gln 20 25 30 Ala Asp Cys Leu Ser Asp Cys Glu Pro Gly AspVal Lys Cys Gln Ser 35 40 45 Tyr Cys Ile Thr Val Pro Ser Pro Asp Glu LysAsn Ile Glu Glu Thr 50 55 60 Thr Lys Cys Cys Leu Pro Pro Ala Pro Arg AlaArg Ala Pro Lys Ala 65 70 75 80 Asp Thr Glu Lys Tyr Thr Val Cys Met AsnGlu Cys Ile Ala Asp Asn 85 90 95 Tyr Trp Lys Ser Val Asp Gly Thr Pro ArgGly Thr Asp Val Pro Asp 100 105 110 Val Lys Ser Lys Ala Ser Glu Ala AlaSer Ser Ala Ala Glu Lys Ala 115 120 125 Thr Ala Thr Gly Thr Ala Ala GluSer Asp Ala Thr Ala Thr Gly Ala 130 135 140 Ser Ala Thr Glu Ser Glu SerGly Ser Asp Ser Ser Ser Glu Glu Thr 145 150 155 160 Gly Ser Ala Ser GlyThr Ala Thr Gly Thr Ala Ala Glu Val Ser Glu 165 170 175 Thr Gly Asn AlaAla Ser Ser Leu Val Gly Gly Val Ser Phe Leu Gly 180 185 190 Leu Val AlaAla Ile Phe Ala Leu 195 200 6 187 PRT Fusarium 6 Met Gly Tyr Tyr Asp AspGlu Gly Ser Phe Arg Asn Gly Gly Ile His 1 5 10 15 Lys Leu Gly Asp LysSer Arg Asp Ile Glu Val Asp Ile Arg Glu Thr 20 25 30 Ser Arg Pro Ala AsnAsp Cys Ala Pro Asn Thr Val Ser Ile Pro Cys 35 40 45 His His Ile Arg LeuGly Asp Phe Leu Met Leu Gln Gly Arg Pro Cys 50 55 60 Gln Val Ile Arg IleSer Thr Ser Ser Ala Thr Gly Gln Tyr Arg Tyr 65 70 75 80 Leu Gly Val AspLeu Phe Thr Lys Gln Leu His Glu Glu Ser Ser Phe 85 90 95 Ile Ser Asn ProAla Pro Ser Val Val Val Gln Ser Met Leu Gly Pro 100 105 110 Val Phe LysGln Tyr Arg Val Leu Asp Met Gln Glu Gly Gln Ile Val 115 120 125 Ala MetThr Glu Thr Gly Asp Val Lys Gln Gly Leu Pro Val Ile Asp 130 135 140 GlnSer Asn Leu Tyr Ser Arg Leu His Asn Ala Phe Glu Ser Gly Arg 145 150 155160 Gly Ser Val Arg Val Leu Val Leu Asn Asp Gly Gly Arg Glu Leu Ala 165170 175 Val Asp Met Lys Val Ile His Gly Ser Arg Leu 180 185 7 30 DNAFusarium 7 gagctcgagg aattcttaca aaccttcaac 30 8 47 DNA Fusarium 8ttaattaagg tacctgaatt taaatggtga agagatagat atccaag 47 9 51 DNA Fusarium9 tcaccattta aattcaggta ccttaattaa attccttgtt ggaagcgtcg a 51 10 42 DNAFusarium 10 tggtatgcat aagcttgaat tcaggtaaac aagatataat tt 42 11 35 DNAFusarium 11 cagtgaattg gcctcgatgg ccgcggccgc gaatt 35 12 35 DNA Fusarium12 aattcgcggc cgcggccatc gaggccaatt cactg 35 13 34 DNA Fusarium 13cacgaaggaa agacgatggc tttcacggtg tctg 34 14 34 DNA Fusarium 14cagacaccgt gaaagccatc gtctttcctt cgtg 34 15 46 DNA Fusarium 15ctatctcttc accatggtac cttaattaaa taccttgttg gaagcg 46 16 46 DNA Fusarium16 cgcttccaac aaggtattta attaaggtac catggtgaag agatag 46 17 30 DNAFusarium 17 atttaaatga tgaggagctc ccttgtgctg 30 18 29 DNA Fusarium 18ttaattaact agagtcgacc cagccgcgc 29 19 45 DNA Fusarium 19 ataagaatgcggccgctagt ttaaacttac aaaccttcaa cagtg 45 20 19 DNA Fusarium 20tagcatctat ctccgtctt 19 21 19 DNA Fusarium 21 gtgtgcagtg acccagaat 19 2242 DNA Fusarium 22 gattgggtcc ctacgtagtt aacactatag gccatcgttt ac 42 2328 DNA Fusarium 23 atttaaatat ggtttcttcg gcattcgc 28 24 28 DNA Fusarium24 ttaattaact attccgacgg aacaaagc 28 25 44 DNA Fusarium 25 ctcttggatatctatctctt caccatggtt tcttcggcat tcgc 44 26 43 DNA Fusarium 26gcgaatgccg aagaaaccat ggtgaagagt agatatccaa gag 43 27 30 DNA Fusarium 27gaatgacttg gttgacgcgt caccagtcac 30 28 26 DNA Fusarium 28 tctagcccagaatactggat caaatc 26 29 39 DNA Fusarium 29 cttaactttg acttgaaaaacatatctgac atttgctcc 39 30 59 DNA Fusarium 30 ggacggcctt ggctagccctccgtgcggcc gccggccggt ctcgcaggat ctgtttaac 59 31 32 DNA Fusarium 31atatcgtgaa gatatgcggc attgatgcca cc 32 32 35 DNA Fusarium 32 ggcggcaataaccggccgaa cattccggat atccc 35 33 32 DNA Fusarium 33 cactgctatcaccaacatgt ttactcaagt cc 32 34 32 DNA Fusarium 34 ggacttgagt aaacatgttggtgatagcag tg 32 35 29 DNA Fusarium 35 gactcatgag gagctccctt gtgctgttc29 36 34 DNA Fusarium 36 tgattaatta acctaaagac atgtcccaat taac 34 37 25DNA Fusarium 37 gcatttaaat tactactgtg atgtg 25 38 25 DNA Fusarium 38gattgatgtg aaacacatgt tgatg 25 39 25 DNA Fusarium 39 cgacccgggaattagagagg ttagg 25 40 28 DNA Fusarium 40 cgtataaccc atggtggact tgtcggac28

What is claimed is:
 1. A method for producing a polypeptide, comprising:(a) cultivating a fungal host cell in a medium for the production of thepolypeptide, wherein the fungal host cell comprises a first nucleic acidsequence encoding the polypeptide operably linked to a second nucleicacid sequence comprising a promoter foreign to the first nucleic acidsequence, wherein the promoter comprises a nucleic acid sequenceselected from the group consisting of (i) nucleotides 1 to 938 of SEQ IDNO:2; (ii) a subsequence of (i) that retains the promoter activity ofnucleotides 1 to 938 of SEQ ID NO:2; and (iii) a nucleic acid sequencethat hybridizes under medium stringency conditions with nucleotides 1 to938 of SEQ ID NO:2; and (b) isolating the polypeptide from thecultivation medium.
 2. The method of claim 1, wherein the promotercomprises nucleotides 1 to 938 of SEQ ID NO:2, or a subsequence thereofthat retains the promoter activity of nucleotides 1 to 938 of SEQ IDNO:2.
 3. The method of claim 1, wherein the promoter comprises thenucleic acid sequence contained in pFAMG which is contained in E. coliNRRL B-30071.
 4. The method of claim 1, wherein the promoter comprises anucleic acid sequence that hybridizes under medium stringency conditionswith nucleotides 1 to 938 of SEQ ID NO:2.
 5. The method of claim 4,wherein the promoter comprises a nucleic acid sequence that hybridizesunder medium-high stringency conditions with nucleotides 1 to 938 of SEQID NO:2.
 6. The method of claim 5, wherein the promoter comprises anucleic acid sequence that hybridizes under high stringency conditionswith nucleotides 1 to 938 of SEQ ID NO:2.
 7. The method of claim 1,wherein the promoter is a hybrid promoter of fragments of two or morepromoters, in which one or more fragments are fragments of nucleotides 1to 938 of SEQ ID NO:2, wherein each fragment contributes to the activityof the hybrid promoter.
 8. The method of claim 7, wherein the hybridpromoter further comprises one or more fragments of a differentpromoter.
 9. The method of claim 1, wherein the promoter is a tandempromoter comprising one or more copies of one or more nucleic acidsequences selected from the group consisting of (a) nucleotides 1 to 938of SEQ ID NO:2; (b) a subsequence of (a) that retains the promoteractivity of nucleotides 1 to 938 of SEQ ID NO:2; and (c) a nucleic acidsequence that hybridizes under medium stringency conditions withnucleotides 1 to 938 of SEQ ID NO:2.
 10. The method of claim 9, whereinthe tandem promoter further comprises one or more different promoters.11. The method of claim 1, wherein the first nucleic acid sequenceencodes a polypeptide heterologous to the fungal host cell.
 12. Themethod of claim 1, wherein the polypeptide is a hormone, enzyme,receptor, antibody or portion thereof, or reporter polypeptide.
 13. Themethod of claim 12, wherein the enzyme is an oxidoreductase,transferase, hydrolase, lyase, isomerase, or ligase.
 14. The method ofclaim 1, wherein the fungal host cell is a filamentous fungal cell oryeast cell.
 15. The method of claim 14, wherein the filamentous fungalcell is an Acremonium, Aspergillus, Fusarium, Humicola, Mucor,Myceliophthora, Neurospora, Penicillium, Thielavia, Tolypocladium, orTrichoderma cell.
 16. The method of claim 14, wherein the yeast cell isa Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces,Schizosaccharomyces, or Yarrowia cell.
 17. The method of claim 14,wherein the filamentous fungal host cell is an Aspergillus cell orFusarium cell.
 18. An isolated promoter sequence comprising a nucleicacid sequence selected from the group consisting of (i) nucleotides 1 to938 of SEQ ID NO:2; (ii) a subsequence of (i) that retains the promoteractivity of nucleotides 1 to 938 of SEQ ID NO:2; and (iii) a nucleicacid sequence that hybridizes under medium stringency conditions withnucleotides 1 to 938 of SEQ ID NO:2.
 19. The promoter sequence of claim18, comprising nucleotides 1 to 938 of SEQ ID NO:2 or a subsequencethereof retaining the promoter activity of nucleotides 1 to 938 of SEQID NO:2.
 20. The isolated promoter sequence of claim 18, comprising thenucleic acid sequence contained in plasmid pFAMG which is contained inE. coli NRRL B-30071.